Synergistic tumor treatment with extended-pk il-2 and therapeutic agents

ABSTRACT

The present invention relates to methods of treating cancer with a combination of extended-PK IL-2 and one or more therapeutic agents, such as a therapeutic antibody. The methods of the invention are applicable across any type of cancer.

GOVERNMENT FUNDING

This invention was made with government support under Grant No.W81XWH-10-1-0291 awarded by the Army Medical Research Command and underGrant No. R01 AI065824 awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

BACKGROUND

Interleukin-2 (IL-2) is a cytokine that induces proliferation ofantigen-activated T cells and stimulates natural killer (NK) cells. Thebiological activity of IL-2 is mediated through a multi-subunit IL-2receptor complex (IL-2R) of three polypeptide subunits that span thecell membrane: p55 (IL-2Rα, the alpha subunit, also known as CD25 inhumans), p75 (IL-2R13, the beta subunit, also known as CD122 in humans)and p64 (IL-2Rγ, the gamma subunit, also known as CD132 in humans). Tcell response to IL-2 depends on a variety of factors, including: (1)the concentration of IL-2; (2) the number of IL-2R molecules on the cellsurface; and (3) the number of IL-2R occupied by IL-2 (i.e., theaffinity of the binding interaction between IL-2 and IL-2R (Smith, “CellGrowth Signal Transduction is Quantal” In Receptor Activation byAntigens, Cytokines, Hormones, and Growth Factors 766:263-271, 1995)).The IL-2:IL-2R complex is internalized upon ligand binding and thedifferent components undergo differential sorting. IL-2Rα is recycled tothe cell surface, while IL-2 associated with the IL-2:IL-2Rβγ complex isrouted to the lysosome and degraded. When administered as an intravenous(i.v.) bolus, IL-2 has a rapid systemic clearance (an initial clearancephase with a half-life of 12.9 minutes followed by a slower clearancephase with a half-life of 85 minutes) (Konrad et al., Cancer Res.50:2009-2017, 1990).

Outcomes of systemic IL-2 administration in cancer patients are far fromideal. While 15 to 20 percent of patients respond objectively tohigh-dose IL-2, the great majority do not, and many suffer severe,life-threatening side effects, including nausea, confusion, hypotension,and septic shock. The severe toxicity associated with IL-2 treatment islargely attributable to the activity of natural killer (NK) cells. NKcells express the intermediate-affinity receptor, IL-2Rβγ_(c), and thusare stimulated at nanomolar concentrations of IL-2, which do in factresult in patient sera during high-dose IL-2 therapy. Attempts to reduceserum concentration, and hence selectively stimulateIL-2Rαβγ_(c)-bearing cells, by reducing dose and adjusting dosingregimen have been attempted, and while less toxic, such treatments werealso less efficacious. Given the toxicity issues associated with highdose IL-2 cancer therapy, numerous groups have attempted to improveanti-cancer efficacy of IL-2 by simultaneously administering therapeuticantibodies. Yet, such efforts have been largely unsuccessful, yieldingno additional or limited clinical benefit compared to IL-2 therapyalone. Accordingly, novel IL-2 therapies are needed to more effectivelycombat various cancers.

SUMMARY

While some attempts have been made to combine IL-2 with therapeuticantibodies to effectively treat various cancers, these efforts have beenlargely unsuccessful. The present invention is based, in part, on thediscovery that prolonging the circulation half-life of IL-2 by attachinga pharmacokinetic modifying group (hereafter referred to as“extended-pharmacokinetic (PK) IL-2”) substantially increases theability of IL-2 to control tumors in various cancer models. Byprolonging circulation half-life, in vivo serum IL-2 concentrations canbe maintained within a therapeutic range, which is not possible withfree IL-2. As discussed infra, the methods of the present inventionallow for synergistic tumor control by combining extended-PK IL-2, withone or more therapeutic agents, such as a therapeutic antibody.

In one aspect, the invention relates to a method for increasing IL-2Rbeta and IL-2R gamma signaling in a lymphocyte in vivo by administeringan agent which stimulates IL2Rβγ_(c), such as an extended-PK interleukin(IL)-2 or a IL-15 superagonist/IL-15Rα complex or an IL-2/IL-2 antibodycomplex, and a therapeutic agent to the cell in an amount effective toincrease IL-2R beta and IL-2R gamma signaling.

In another aspect, the invention relates to a method for treating cancerin a subject by administering an extended-PK IL-2, and a therapeuticagent in an amount effective to treat cancer. The cancer to be treatedcan be, e.g., melanoma, colon cancer, breast cancer, renal cancer,testicular cancer, ovarian cancer, prostate cancer, cancer of the smallintestine, cancer of the esophagus, cervical cancer, lung cancer,lymphoma, and leukemia.

In another aspect, the invention relates to a method for treating cancerand reducing vascular leak syndrome associated with IL-2 therapy in asubject by administering an extended-PK IL-2, and a therapeutic antibodyin an amount effective to treat cancer and reduce vascular leak syndromeassociated with IL-2 therapy in the subject. In another aspect, theinvention relates to a method for treating cancer and reducing pulmonaryedema associated with IL-2 therapy in a subject by administering anextended-PK IL-2, and a therapeutic antibody in an amount effective totreat cancer and reduce pulmonary edema associated with IL-2 therapy inthe subject.

In another aspect, the invention relates to a method of inhibiting thegrowth and/or proliferation of tumor cells in a subject by administeringan extended-PK IL-2 and a therapeutic antibody in an amount effective toinhibit growth and/or proliferation of tumor cells in the subject. Inone aspect, the methods of the invention result in a reduction in tumorsize in the subject, for example, by at least 30%, at least 50%, atleast 80%, or at least 90%. In one embodiment, the extended-PK IL-2 andtherapeutic agent reduces tumor size to a greater extent than achievedby a combination of IL-2 and a therapeutic antibody. In another aspect,the treatment according to the invention increases the recruitment oflymphocytes to the periphery of a tumor. In one embodiment, such methodsinhibit primary tumor metastasis. In yet another aspect, methods of theinvention prolong the survival of a subject with a tumor, such as amouse model of cancer, by, e.g., 25 days or more.

In another aspect, the invention relates to a method of stimulating Tcells and/or NK cells in a subject by administering an extended-PKinterleukin (IL)-2, and a therapeutic agent in an amount effective tostimulate T cells and/or NK cells in a subject. In one embodiment, thestimulation of T cells and/or NK cells leads to enhancedantibody-dependent cell-mediated cytotoxicity (ADCC) and/or cytotoxic Tlymphocyte (CTL) responses. In another embodiment, the stimulation of Tcells and/or NK cells leads to an increased number of CD8+ T cells in asubject.

The extended-PK IL-2 of the methods described above can be in the formof a fusion protein, such as an IL-2 moiety fused to an immunoglobulinfragment such as Fc, human serum albumin, or Fn3. Alternatively, theextended-PK IL-2 is conjugated to a non-protein polymer, such as PEG.When the IL-2 moiety is fused to an Fc domain, the Fc domain may bemutated to reduce binding to Fcγ receptors, complement proteins, orboth, i.e., to reduce effector function. In other embodiments, thefusion protein comprises a monomer of one IL-2 moiety linked to an Fcdomain as a heterodimer, or a dimer of two IL-2 moieties linked to an Fcdomain as a heterodimer. In other embodiments, IL-2 is mutated such thatit has higher affinity for the IL-2R alpha receptor compared tounmodified IL-2.

The therapeutic agents to be used in combination with the extended-PKIL-2 of the methods described above can be, e.g., a therapeuticantibody, a therapeutic protein, a small molecule, an antigen, or apopulation of cells. The extended-PK IL-2 and the therapeutic agent areadministered simultaneously or sequentially. In one embodiment, theextended-PK IL-2 and the therapeutic agent are administered within threedays of each other. In another embodiment, one or more additionaltherapeutic agents are added to the combination of extended-PK IL-2 andtherapeutic agent. Such agents can be, e.g., a cytokine, achemotherapeutic agent, and/or a population of cytotoxic T cells.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1 depicts the sequences of high affinity CD25-binding mouse IL-2mutants generated by error prone PCR and yeast surface display. mIL-2depicts the sequence of murine IL-2. The locations of mutations in theIL-2 mutants are shown. The mutants with names preceded by “QQ” arethose in which putative IL-2Rβ-binding mutations were reverted back towild-type residues by site directed mutagenesis.

FIG. 2 is a series of graphs depicting the affinity of the indicatedIL-2 mutants for soluble murine CD25. The equilibrium dissociationconstant was determined as described in Chao et al. (Nat Protocols 2006;1(2):755-768). Diamonds indicate wild-type murine IL-2; squares indicateIL-2 6.2-10; triangles indicate IL-2 mutants in which putativeIL-2Rβ-binding mutations were reverted back to wild-type residues.

FIG. 3 is a three dimensional model of murine IL-2 bound to murine CD25generated using SWISS-MODEL (Schwede et al., Nucleic Acids Research2003; 31:3381-5). Residues E76, H82, and Q121 are in close contact withCD25.

FIG. 4 is a series of flow cytometry histograms showing the display ofE76A IL-2 on the surface of yeast (as determined by anti-HA andanti-c-myc staining), its lack of detectable binding to soluble murineCD25 at 50 nM, and its proper folding (as detected by anti-IL-2antibodies S4B6, JES6-1A12, and JESA-5H4 before and after thermaldenaturation).

FIG. 5 is a schematic of D265AFc/IL-2 (hereafter referred to as“Fc/IL-2”). IL-2 is monovalent and has a K_(D) of about 50 nM for mouseCD25. The beta half-life of Fc/IL-2 is about 15 hours.

FIG. 6 is a series of graphs depicting the viability of CTLL-2 cellsstimulated with the indicated Fc/IL-2 and mutants. CTLL-2 cells werestimulated with Fc/IL-2, Fc/QQ6210, Fc/E76A, or Fc/E76G for 30 minutes,then resuspended in cytokine-free medium. At indicated times aftercytokine withdrawal, culture aliquots were used to measure cultureviability as determined by cellular ATP content, which was assayedthrough stimulation of ATP-dependent luciferase activity using theCellTiter-Glo Luminescent Viability Assay (Promega).

FIG. 7 is a photograph of spleens isolated from C57BL/6 mice (n=3/group)injected intravenously with PBS or 25 μg Fc/IL-2, Fc/QQ6210, or Fc/E76G.Spleens were isolated 4 days after treatment. Two representative spleensper group are shown.

FIG. 8 is a series of graphs depicting various lymphocyte populations inspleens isolated from mice treated under the conditions described inFIG. 7. Populations of cell types are as indicated. CD3+CD8+ depictsCD8+ T cells, and CD3−NK1.1+ depicts natural killer (NK) cells. Errorbars represent standard deviation for measurements of three samples.

FIG. 9 is a graph depicting total weight change (grams), which is usedas a proxy for toxicity, in C57BL/6 mice injected with PBS, Fc/IL-2,Fc/QQ6210, or Fc/E76G as described in FIG. 7.

FIG. 10 is a graph depicting total lung wet weight (grams), which isused as an indicator of pulmonary edema and vascular leak syndrome.C57BL/6 mice injected with PBS, Fc/IL-2, Fc/QQ6210, or Fc/E76G asdescribed in FIG. 7.

FIG. 11 is a series of graphs depicting the anti-tumor effects ofFc/IL-2 and TA99 antibody. C57BL/6 mice (n=5/group) were injectedsubcutaneously with 10⁶ B16-F10 melanoma cells. Six days after tumorinoculation mice were injected intravenously with PBS, 6 μg IL-2, 25 μgFc/IL-2, 100 μg TA99, IL-2 (6 μg)+TA99 (100 μg), or Fc/IL-2 (25 μg)+TA99(100 μg). Subsequent doses were administered every 6 days. Eachindividual line represents one mouse and inverted triangles represent aninjection of the indicated regimen.

FIG. 12 is a series of graphs depicting the average tumor volume of eachtreatment group shown in FIG. 11. Bars represent standard deviation.

FIG. 13 is a graph depicting the number of days it took for tumors ineach treatment group shown in FIG. 11 to reach an area >100 mm². Tumorarea was calculated as l×w, wherein l=longest dimension of the tumor andw=longest dimension perpendicular to 1.

FIG. 14 is a series of graphs depicting animal weight (grams) in micetreated as described for FIG. 11. Shown is animal weight normalized toinitial weight at time of tumor inoculation.

FIG. 15 is a graph depicting the time for tumor volume to double twicefrom its value at initial treatment based on the degree of timeseparation between Fc/IL-2 and TA99 injection. C57BL/6 mice wereinjected subcutaneously with 106 B16-F10 melanoma cells. Six days aftertumor inoculation, mice were injected intravenously with PBS (dashedlines), or a single dose each of 25 μg Fc/IL-2 and 100 μg TA99(diamonds). The y-axis represents the time for tumor volume to doubletwice from its value at the initiation of treatment, V₀. The x-axisrepresents the time separation between Fc/IL-2 and TA99 injection, wherethe time of Fc/IL-2 injection has been set as the reference, t=0. Datashown for two independent experiments.

FIG. 16 is a series of photomicrographs depicting the recruitment oflymphocytes to the periphery of tumors. Hematoxylin and eosin stainedsections of subcutaneous B16-F10 tumors four days after a single dose ofPBS or 25 μg Fc/IL-2 and 100 μg TA99 at 10× magnification. Images arerepresentative of two independent experiments.

FIG. 17 is a series of graphs depicting the protection conferred byFc/IL-2 and TA99 combination therapy against secondary tumor challenge.C57BL/6 mice bearing B16-F10 tumors were treated with five doses of 25μg Fc/IL-2 and 100 μg TA99 (n=3). These mice were injectedsubcutaneously with 10⁵ B16-F10 melanoma cells in the opposite flanktwelve days after the last treatment. Untreated naive C57BL/6 mice (n=2)were also injected subcutaneously with 10⁵ B16-F10 melanoma cells. Eachindividual line represents one mouse and inverted triangles represent are-challenge with 10⁵ B16-F10 melanoma cells.

FIG. 18 is a series of graphs demonstrating that CD25 binding affinityis required for maximal Fc/IL-2+TA99 combination therapy. C57BL/6 mice(n=5 mice/group) were injected subcutaneously with 10⁶ B16-F10 melanomacells. Six days after tumor inoculation, mice were injectedintravenously with 25 μg Fc/QQ6210 or Fc/E76G, alone or with 100 μgTA99. Subsequent doses were administered every 6 days. Each individualline represents one mouse and inverted triangles represent an injectionof the indicated regimen.

FIG. 19 depicts the fluorescence activated cell sorting (FACS)-mediatedconfirmation of NK cell or CD8+ cell depletion by anti-NK1.1 oranti-CD8a antibody, respectively. C57BL/6 mice (n=1 mouse/group) wereinjected subcutaneously with 10⁶ B16-F10 melanoma cells. Four days aftertumor inoculation, mice were injected intraperitoneally with 400 μganti-NK1.1 or 400 μg anti-CD8a antibody. Two days after antibodyinjection, single-cell suspensions were prepared from spleens andstained with calcein violet AM, PE-conjugated anti-CD3, andAPC-conjugated anti-NK1.1 or Alexa Fluor 647-conjugated anti-CD8a.Untreated controls did not receive tumor inoculation or antibodyinjection. Cells were gated by forward scatter and calcein violet AM.The internal box in the panels on the left reflect NK cells, and theinternal box in the panels on the right reflect CD8+ T cells.

FIG. 20 is a series of graphs demonstrating that NK and CD8+ T cellscontribute to the anti-tumor effects of Fc/IL-2+TA99 combinationtherapy. C57BL/6 mice (n=5 mice per group) were injected subcutaneouslywith 10⁶ B16-F10 melanoma cells. Four days after tumor inoculation, micewere injected intraperitoneally with 400 μg anti-NK1.1 or 400 μganti-CD8a antibody; subsequent doses were administered every four days.Six days after tumor inoculation, mice were injected intravenously withPBS, 25 μg Fc/IL-2 and 100 μg TA99; subsequent doses were administeredevery 6 days. Each individual line represents one mouse and invertedtriangles represent an injection of Fc/IL-2+TA99.

FIG. 21 is a graph depicting the effects of Fc/IL-2 and sm3E anti-CEAantibody in controlling tumor growth in a mouse model of colon cancer.MC38-CEA cells (1×10⁶), a colon cancer cell line that was engineered totransgenically express CEA, were injected into the flank of four C57BL/6 J mice to induce tumor establishment. Fc-IL-2 was injected at adosage of 25 μg/mouse retroorbitally. Sm3E anti-CEA antibody wasinjected at a dosage of 200 μg/mouse retroorbitally. Tumor volume wasassessed as described in Example 9.

DETAILED DESCRIPTION

In one aspect, the present invention relates to a method of treatingcancer comprising administering an extended-PK IL-2 and one or moretherapeutic agents, such as a therapeutic antibody, with or without oneor more additional agents, to a subject in need thereof in an amountsufficient to treat cancer, e.g., to reduce the tumor size and growth inthe subject. In another aspect, the methods of the present inventionprolong survival of subjects with cancer. In other aspects, extended-PKIL-2 and one or more therapeutic agents synergizes to exert potent tumorgrowth suppression.

DEFINITIONS

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified. In the case of direct conflict with aterm used in a parent provisional patent application, the term used inthe instant specification shall control.

“Amino acid” refers to naturally occurring and synthetic amino acids, aswell as amino acid analogs and amino acid mimetics that function in amanner similar to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, i.e., an α carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs have modified R groups (e.g., norleucine) or modified peptidebackbones, but retain the same basic chemical structure as a naturallyoccurring amino acid. Amino acid mimetics refers to chemical compoundsthat have a structure that is different from the general chemicalstructure of an amino acid, but that function in a manner similar to anaturally occurring amino acid.

Amino acids can be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,can be referred to by their commonly accepted single-letter codes.

An “amino acid substitution” refers to the replacement of at least oneexisting amino acid residue in a predetermined amino acid sequence (anamino acid sequence of a starting polypeptide) with a second, different“replacement” amino acid residue. An “amino acid insertion” refers tothe incorporation of at least one additional amino acid into apredetermined amino acid sequence. While the insertion will usuallyconsist of the insertion of one or two amino acid residues, the presentlarger “peptide insertions,” can be made, e.g. insertion of about threeto about five or even up to about ten, fifteen, or twenty amino acidresidues. The inserted residue(s) may be naturally occurring ornon-naturally occurring as disclosed above. An “amino acid deletion”refers to the removal of at least one amino acid residue from apredetermined amino acid sequence.

“Polypeptide,” “peptide”, and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues. The terms apply to aminoacid polymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid and are metabolized in a mannersimilar to naturally occurring nucleotides. Unless otherwise indicated,a particular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences and as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions canbe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991;Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985); and Cassol et al.,1992; Rossolini et al., Mol. Cell. Probes 8:91-98, 1994). For arginineand leucine, modifications at the second base can also be conservative.The term nucleic acid is used interchangeably with gene, cDNA, and mRNAencoded by a gene. Polynucleotides of the present invention can becomposed of any polyribonucleotide or polydeoxyribonucleotide, which canbe unmodified RNA or DNA or modified RNA or DNA. For example,polynucleotides can be composed of single- and double-stranded DNA, DNAthat is a mixture of single- and double-stranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatcan be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. In addition, the polynucleotidecan be composed of triple-stranded regions comprising RNA or DNA or bothRNA and DNA. A polynucleotide can also contain one or more modifiedbases or DNA or RNA backbones modified for stability or for otherreasons. “Modified” bases include, for example, tritylated bases andunusual bases such as inosine. A variety of modifications can be made toDNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically,or metabolically modified forms.

As used herein, the term “PK” is an acronym for “pharmacokinetic” andencompasses properties of a compound including, by way of example,absorption, distribution, metabolism, and elimination by a subject. Asused herein, an “extended-PK group” refers to a protein, peptide, ormoiety that increases the circulation half-life of a biologically activemolecule when fused to or administered together with the biologicallyactive molecule. Examples of an extended-PK group include PEG, humanserum albumin (HSA) binders (as disclosed in U.S. Publication Nos.2005/0287153 and 2007/0003549, PCT Publication Nos. WO 2009/083804 andWO 2009/133208, and SABA molecules as described in US2012/094909), humanserum albumin, Fc or Fc fragments and variants thereof, and sugars(e.g., sialic acid). Other exemplary extended-PK groups are disclosed inKontermann et al., Current Opinion in Biotechnology 2011; 22:868-876,which is herein incorporated by reference in its entirety. As usedherein, an “extended-PK IL-2” refers to an IL-2 moiety in combinationwith an extended-PK group. In one embodiment, the extended-PK IL-2 is afusion protein in which an IL-2 moiety is linked or fused to anextended-PK group. An exemplary fusion protein is a Fc/IL-2 fusion inwhich one or more IL-2 moieties are linked to an immunoglobulin Fcdomain (e.g., an IgG1 Fc domain).

The term “extended-PK IL-2” is also intended to encompass IL-2 mutantswith mutations in one or more amino acid residues that enhances theaffinity of IL-2 for one or more of its receptors, for example, CD25. Inone embodiment, the IL-2 moiety of extended-PK IL-2 is wild-type IL-2.In another embodiment, the IL-2 moiety is a mutant IL-2 which exhibitsgreater affinity for CD25 than wild-type IL2, such as one of the IL-2mutants depicted in FIG. 1. When a particular type of extended-PK groupis indicated, such as PEG-IL-2, it should be understood that thisencompasses both PEG conjugated to a wild-type IL-2 moiety or a PEGconjugated to a mutant IL-2 moiety.

In certain aspects, the extended-PK IL-2 of the invention can employ oneor more “linker domains,” such as polypeptide linkers. As used herein,the term “linker domain” refers to a sequence which connects two or moredomains (e.g., the PK moiety and IL-2) in a linear sequence. As usedherein, the term “polypeptide linker” refers to a peptide or polypeptidesequence (e.g., a synthetic peptide or polypeptide sequence) whichconnects two or more domains in a linear amino acid sequence of apolypeptide chain. For example, polypeptide linkers may be used toconnect an IL-2 moiety to an Fc domain. Preferably, such polypeptidelinkers can provide flexibility to the polypeptide molecule. In certainembodiments the polypeptide linker is used to connect (e.g., geneticallyfuse) one or more Fc domains and/or IL-2.

As used herein, the terms “linked,” “fused”, or “fusion”, are usedinterchangeably. These terms refer to the joining together of two moreelements or components or domains, by whatever means including chemicalconjugation or recombinant means. Methods of chemical conjugation (e.g.,using heterobifunctional crosslinking agents) are known in the art.

As used herein, the term “Fc region” shall be defined as the portion ofa native immunoglobulin formed by the respective Fc domains (or Fcmoieties) of its two heavy chains. As used herein, the term “Fc domain”refers to a portion of a single immunoglobulin (Ig) heavy chain whereinthe Fc domain does not comprise an Fv domain. As such, Fc domain canalso be referred to as “Ig” or “IgG.” In some embodiments, an Fc domainbegins in the hinge region just upstream of the papain cleavage site andending at the C-terminus of the antibody. Accordingly, a complete Fcdomain comprises at least a hinge domain, a CH2 domain, and a CH3domain. In certain embodiments, an Fc domain comprises at least one of:a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragmentthereof. In other embodiments, an Fc domain comprises a complete Fcdomain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In oneembodiment, an Fc domain comprises a hinge domain (or portion thereof)fused to a CH3 domain (or portion thereof). In another embodiment, an Fcdomain comprises a CH2 domain (or portion thereof) fused to a CH3 domain(or portion thereof). In another embodiment, an Fc domain consists of aCH3 domain or portion thereof. In another embodiment, an Fc domainconsists of a hinge domain (or portion thereof) and a CH3 domain (orportion thereof). In another embodiment, an Fc domain consists of a CH2domain (or portion thereof) and a CH3 domain. In another embodiment, anFc domain consists of a hinge domain (or portion thereof) and a CH2domain (or portion thereof). In one embodiment, an Fc domain lacks atleast a portion of a CH2 domain (e.g., all or part of a CH2 domain). AnFc domain herein generally refers to a polypeptide comprising all orpart of the Fc domain of an immunoglobulin heavy-chain. This includes,but is not limited to, polypeptides comprising the entire CH1, hinge,CH2, and/or CH3 domains as well as fragments of such peptides comprisingonly, e.g., the hinge, CH2, and CH3 domain. The Fc domain may be derivedfrom an immunoglobulin of any species and/or any subtype, including, butnot limited to, a human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgMantibody. A human IgG1 constant region can be found at Uniprot P01857and in Table 5 (i.e., SEQ ID NO: 33). The Fc domain of human IgG1 can befound in Table 5 (i.e., SEQ ID NO: 34). The Fc domain encompasses nativeFc and Fc variant molecules. As with Fc variants and native Fc's, theterm Fc domain includes molecules in monomeric or multimeric form,whether digested from whole antibody or produced by other means. Theassignment of amino acid residue numbers to an Fc domain is inaccordance with the definitions of Kabat. See, e.g., Sequences ofProteins of Immunological Interest (Table of Contents, Introduction andConstant Region Sequences sections), 5th edition, Bethesda, Md.:NIH vol.1:647-723 (1991); Kabat et al., “Introduction” Sequences of Proteins ofImmunological Interest, US Dept of Health and Human Services, NIH, 5thedition, Bethesda, Md. vol. 1:xiii-xcvi (1991); Chothia & Lesk, J. Mol.Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989),each of which is herein incorporated by reference for all purposes.

As set forth herein, it will be understood by one of ordinary skill inthe art that any Fc domain may be modified such that it varies in aminoacid sequence from the native Fc domain of a naturally occurringimmunoglobulin molecule. In certain exemplary embodiments, the Fc domainhas reduced effector function (e.g., FcγR binding).

The Fc domains of a polypeptide of the invention may be derived fromdifferent immunoglobulin molecules. For example, an Fc domain of apolypeptide may comprise a CH2 and/or CH3 domain derived from an IgG1molecule and a hinge region derived from an IgG3 molecule. In anotherexample, an Fc domain can comprise a chimeric hinge region derived, inpart, from an IgG1 molecule and, in part, from an IgG3 molecule. Inanother example, an Fc domain can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

A polypeptide or amino acid sequence “derived from” a designatedpolypeptide or protein refers to the origin of the polypeptide.Preferably, the polypeptide or amino acid sequence which is derived froma particular sequence has an amino acid sequence that is essentiallyidentical to that sequence or a portion thereof, wherein the portionconsists of at least 10-20 amino acids, preferably at least 20-30 aminoacids, more preferably at least 30-50 amino acids, or which is otherwiseidentifiable to one of ordinary skill in the art as having its origin inthe sequence.

Polypeptides derived from another peptide may have one or more mutationsrelative to the starting polypeptide, e.g., one or more amino acidresidues which have been substituted with another amino acid residue orwhich has one or more amino acid residue insertions or deletions.

A polypeptide can comprise an amino acid sequence which is not naturallyoccurring. Such variants necessarily have less than 100% sequenceidentity or similarity with the starting IL-2 molecule. In a preferredembodiment, the variant will have an amino acid sequence from about 75%to less than 100% amino acid sequence identity or similarity with theamino acid sequence of the starting polypeptide, more preferably fromabout 80% to less than 100%, more preferably from about 85% to less than100%, more preferably from about 90% to less than 100% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%) and most preferably from about 95% toless than 100%, e.g., over the length of the variant molecule.

In one embodiment, there is one amino acid difference between a startingpolypeptide sequence and the sequence derived therefrom. Identity orsimilarity with respect to this sequence is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical (i.e., same residue) with the starting amino acid residues,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity.

In one embodiment, a polypeptide of the invention consists of, consistsessentially of, or comprises an amino acid sequence selected from SEQ IDNOs: 2, 4, 6, 8, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32. In anembodiment, a polypeptide includes an amino acid sequence at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selectedfrom SEQ ID NOs: 2, 4, 6, 8, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and32. In an embodiment, a polypeptide includes a contiguous amino acidsequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguousamino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, and 32. In an embodiment, a polypeptideincludes an amino acid sequence having at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or500 (or any integer within these numbers) contiguous amino acids of anamino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, and 32.

In an embodiment, the peptides of the invention are encoded by anucleotide sequence. Nucleotide sequences of the invention can be usefulfor a number of applications, including: cloning, gene therapy, proteinexpression and purification, mutation introduction, DNA vaccination of ahost in need thereof, antibody generation for, e.g., passiveimmunization, PCR, primer and probe generation, and the like. In anembodiment, the nucleotide sequence of the invention comprises, consistsof, or consists essentially of, a nucleotide sequence selected from SEQID NOs: 1, 3, 5, 7, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31. Inan embodiment, a nucleotide sequence includes a nucleotide sequence atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequenceset forth in SEQ ID NOs: 1, 3, 5, 7, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, and 31. In an embodiment, a nucleotide sequence includes acontiguous nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a contiguous nucleotide sequence set forth in SEQ ID NOs:1, 3, 5, 7, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31. In anembodiment, a nucleotide sequence includes a nucleotide sequence havingat least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 200, 300, 400, or 500 (or any integer within these numbers)contiguous nucleotides of a nucleotide sequence set forth in SEQ ID NOs:1, 3, 5, 7, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31.

It will also be understood by one of ordinary skill in the art that theextended-PK IL-2 of the invention may be altered such that they vary insequence from the naturally occurring or native sequences from whichthey were derived, while retaining the desirable activity of the nativesequences. For example, nucleotide or amino acid substitutions leadingto conservative substitutions or changes at “non-essential” amino acidresidues may be made. Mutations may be introduced by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis.

The IL-2 and Fc molecules of the invention may comprise conservativeamino acid substitutions at one or more amino acid residues, e.g., atessential or non-essential amino acid residues. A “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart, including basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a nonessential amino acid residue in a bindingpolypeptide is preferably replaced with another amino acid residue fromthe same side chain family. In another embodiment, a string of aminoacids can be replaced with a structurally similar string that differs inorder and/or composition of side chain family members. Alternatively, inanother embodiment, mutations may be introduced randomly along all orpart of a coding sequence, such as by saturation mutagenesis, and theresultant mutants can be incorporated into binding polypeptides of theinvention and screened for their ability to bind to the desired target.

The term “ameliorating” refers to any therapeutically beneficial resultin the treatment of a disease state, e.g., cancer, includingprophylaxis, lessening in the severity or progression, remission, orcure thereof.

The term “in situ” refers to processes that occur in a living cellgrowing separate from a living organism, e.g., growing in tissueculture.

The term “in vivo” refers to processes that occur in a living organism.

The term “mammal” or “subject” or “patient” as used herein includes bothhumans and non-humans and include but is not limited to humans,non-human primates, canines, felines, murines, bovines, equines, andporcines.

The term percent “identity,” in the context of two or more nucleic acidor polypeptide sequences, refer to two or more sequences or subsequencesthat have a specified percentage of nucleotides or amino acid residuesthat are the same, when compared and aligned for maximum correspondence,as measured using one of the sequence comparison algorithms describedbelow (e.g., BLASTP and BLASTN or other algorithms available to personsof skill) or by visual inspection. Depending on the application, thepercent “identity” can exist over a region of the sequence beingcompared, e.g., over a functional domain, or, alternatively, exist overthe full length of the two sequences to be compared.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

As used herein, the term “gly-ser polypeptide linker” refers to apeptide that consists of glycine and serine residues. An exemplarygly-ser polypeptide linker comprises the amino acid sequenceSer(Gly₄Ser)n. In one embodiment, n=1. In one embodiment, n=2. Inanother embodiment, n=3, i.e., Ser(Gly₄Ser)3. In another embodiment,n=4, i.e., Ser(Gly₄Ser)4. In another embodiment, n=5. In yet anotherembodiment, n=6. In another embodiment, n=7. In yet another embodiment,n=8. In another embodiment, n=9. In yet another embodiment, n=10.Another exemplary gly-ser polypeptide linker comprises the amino acidsequence (Gly₄Ser)n. In one embodiment, n=1. In one embodiment, n=2. Ina preferred embodiment, n=3. In another embodiment, n=4. In anotherembodiment, n=5. In yet another embodiment, n=6. Another exemplarygly-ser polypeptide linker comprises the amino acid sequence (Gly₃Ser)n.In one embodiment, n=1. In one embodiment, n=2. In a preferredembodiment, n=3. In another embodiment, n=4. In another embodiment, n=5.In yet another embodiment, n=6.

As used herein, the terms “linked,” “fused”, or “fusion” are usedinterchangeably. These terms refer to the joining together of two moreelements or components or domains, by whatever means including chemicalconjugation or recombinant means. Methods of chemical conjugation (e.g.,using heterobifunctional cros slinking agents) are known in the art.

As used herein, “half-life” refers to the time taken for the serum orplasma concentration of a polypeptide to reduce by 50%, in vivo, forexample due to degradation and/or clearance or sequestration by naturalmechanisms. The extended-PK IL-2 of the present invention is stabilizedin vivo and its half-life increased by, e.g., fusion to an Fc region,through PEGylation, or by binding to serum albumin molecules (e.g.,human serum albumin) which resist degradation and/or clearance orsequestration. The half-life can be determined in any manner known perse, such as by pharmacokinetic analysis. Suitable techniques will beclear to the person skilled in the art, and may for example generallyinvolve the steps of suitably administering a suitable dose of the aminoacid sequence or compound of the invention to a subject; collectingblood samples or other samples from said subject at regular intervals;determining the level or concentration of the amino acid sequence orcompound of the invention in said blood sample; and calculating, from (aplot of) the data thus obtained, the time until the level orconcentration of the amino acid sequence or compound of the inventionhas been reduced by 50% compared to the initial level upon dosing.Further details are provided in, e.g., standard handbooks, such asKenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbookfor Pharmacists and in Peters et al., Pharmacokinetic Analysis: APractical Approach (1996). Reference is also made to Gibaldi, M. et al.,Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).

A “therapeutic antibody” is an antibody, fragment of an antibody, orconstruct that is derived from an antibody, and can bind to acell-surface antigen on a target cell to cause a therapeutic effect.Such antibodies can be chimeric, humanized or fully human antibodies.Methods are known in the art for producing such antibodies. Suchantibodies include single chain Fc fragments of antibodies, minibodiesand diabodies. Any of the therapeutic antibodies known in the art to beuseful for cancer therapy can be used in combination therapy withextended-PK IL-2 of the present invention. Therapeutic antibodies may bemonoclonal antibodies or polyclonal antibodies. In preferredembodiments, the therapeutic antibodies target cancer antigens.

As used herein, “cancer antigen” refers to (i) tumor-specific antigens,(ii) tumor-associated antigens, (iii) cells that express tumor-specificantigens, (iv) cells that express tumor-associated antigens, (v)embryonic antigens on tumors, (vi) autologous tumor cells, (vii)tumor-specific membrane antigens, (viii) tumor-associated membraneantigens, (ix) growth factor receptors, (x) growth factor ligands, and(xi) any other type of antigen or antigen-presenting cell or materialthat is associated with a cancer.

As used herein, a “small molecule” is a molecule with a molecular weightbelow about 500 Daltons.

As used herein, “therapeutic protein” refers to any polypeptide,protein, protein variant, fusion protein and/or fragment thereof whichmay be administered to a subject as a medicament. An exemplarytherapeutic protein is an interleukin, e.g., IL-7.

As used herein, “synergy” or “synergistic effect” with regard to aneffect produced by two or more individual components refers to aphenomenon in which the total effect produced by these components, whenutilized in combination, is greater than the sum of the individualeffects of each component acting alone.

The term “sufficient amount” or “amount sufficient to” means an amountsufficient to produce a desired effect, e.g., an amount sufficient toreduce the size of a tumor.

The term “therapeutically effective amount” is an amount that iseffective to ameliorate a symptom of a disease. A therapeuticallyeffective amount can be a “prophylactically effective amount” asprophylaxis can be considered therapy.

As used herein, “combination therapy” embraces administration of eachagent or therapy in a sequential manner in a regiment that will providebeneficial effects of the combination, and co-administration of theseagents or therapies in a substantially simultaneous manner, such as in asingle capsule having a fixed ratio of these active agents or inmultiple, separate capsules for each agent. Combination therapy alsoincludes combinations where individual elements may be administered atdifferent times and/or by different routes but which act in combinationto provide a beneficial effect by co-action or pharmacokinetic andpharmacodynamics effect of each agent or tumor treatment approaches ofthe combination therapy. As used herein, “about” will be understood bypersons of ordinary skill and will vary to some extent depending on thecontext in which it is used. If there are uses of the term which are notclear to persons of ordinary skill given the context in which it isused, “about” will mean up to plus or minus 10% of the particular value.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

Overview

In one aspect, the present invention relates to methods of treatingcancer in a subject comprising administering an extended-pharmacokinetic(PK) interleukin (IL)-2, and a therapeutic agent, e.g., a therapeuticantibody, in an amount effective to treat cancer. In some aspects, theextended-PK IL-2 comprises a fusion protein, wherein the fusioncomprises an IL-2 moiety and another moiety, such as an immunoglobulinfragment, a conjugated non-protein polymer, human serum albumin, or Fn3.In a preferred embodiment, the non-IL-2 moiety of the fusion is an Fcdomain, preferably the Fc region from human IgG1. In some aspects, theFc domain is mutated so as to reduce binding to Fcγ receptors,complement proteins, or both. Such Fc domains exhibit reduced effectorfunctions. One such mutant is an Fc region in which the aspartic acidresidue at position 265 is mutated to alanine. In some embodiments, thefusion protein comprises a monomer of one IL-2 moiety linked to an Fcdomain as a heterodimer. In other embodiments, the fusion proteincomprises a dimer of two IL-2 moieties linked to an Fc domain as aheterodimer (i.e., bivalent IL-2). The invention also relates to novelIL-2 mutants that exhibit high affinity binding to the IL-2R alphareceptor (i.e., CD25). These IL-2 mutants are suited for use in themethods of the present invention.

The therapeutic agent to be used in conjunction with extended-PK IL-2can be, for example, a therapeutic antibody, a therapeutic protein, asmall molecule, an antigen, or a population of cells (e.g., a populationof ex vivo expanded CD8+ T cells). In a preferred embodiment, thetherapeutic agent is a therapeutic antibody. In some embodiments, morethan one therapeutic agent can be included in the combination therapywith extended PK-IL2. Extended-PK IL-2 and the therapeutic agent can beadministered simultaneously or sequentially. In some embodiments, theextended-PK IL-2 and the therapeutic agent are administered within threedays of each other.

IL-2 signals through a receptor complex consisting of IL-2Rα, IL-2Rβ,and IL-2Rγ. The signaling events resulting from IL-2 stimulation areinitiated by ligand-induced oligomerization of these receptor subunits.In particular, the α, β, and γ_(c) chains recognize distinct sites onIL-2 and associate with IL-2 in a stepwise manner, with IL-2Rα bindingIL-2 leading to subsequent recruitment of IL-2Rβ and γ_(c) anddownstream signaling. This signaling promotes proliferation and cellsurvival. Accordingly, in one embodiment, the present invention relatesto a method of increasing IL-2Rβ and IL-2Rγ gamma signaling in alymphocyte by administering a therapeutic agent or agents in an amounteffective for stimulating the IL-2Rβγ complex. In one embodiment, thetherapeutic agent is an extended-PK IL-2.

In another embodiment, the therapeutic agent is a IL-15superagonist/IL-15Rα complex (IL-15 shares IL-2Rβγ subunits with IL-2for downstream signaling), such as that disclosed in Rubinstein et al.(PNAS, 2006; 103:9166-71). The IL-15 superagonist/IL-15Rα complex alonemay have prolonged serum half-life given its bulky size, therebyallowing for prolonged IL-2Rβγ signaling. Indeed, as described in theExamples, extended-PK IL-2 that does not bind to IL-2Rα exerted atherapeutic benefit against tumor growth, suggesting that an agent(s)that promotes IL-2Rβγ signaling will also likely have a therapeuticeffect in the treatment of cancer. As shown in FIG. 18, extended-PK IL-2variants lacking CD25 binding still exert some therapeutic effect,albeit less than when capable of CD25 binding.

In yet another embodiment, the therapeutic agent is an IL-2/anti-IL-2antibody complex that specifically activates IL-2Rβγ signaling (such asthat disclosed in Krieg et al. (PNAS 2010; 107:11906-11)) in combinationwith another therapeutic antibody. While CD25 binding is not requiredfor the therapeutic effects of the therapeutic agents of the presentinvention, in preferred embodiments, the agent(s) have CD25 bindingability.

The methods of the invention are also useful for reducing tumor growthor size in a subject. In one embodiment, extended-PK IL-2 in combinationwith one or more therapeutic antibodies, with or without one or moreadditional agents, is administered to a subject with established tumorsin an amount sufficient to reduce tumor growth or size in the subject.In some embodiments, tumor growth or size is decreased by about 30%,about 50%, about 70%, or about 90% compared to the size of the tumorprior to the combination therapy.

In another embodiment, the methods of the present invention are usefulfor inhibiting the growth and/or proliferation of tumor cells in asubject by administering an extended-PK IL-2 and a therapeutic antibodyin an amount effective to inhibit growth and/or proliferation of tumorcells in the subject. In one embodiment, the combination therapy ofextended-PK IL-2 and a therapeutic antibody results in enhanced orincreased suppression of tumor or tumor cell growth as compared tounmodified IL-2 (e.g., recombinant human IL-2).

In other embodiments, the methods of the present invention are usefulfor prolonging the survival of a subject with a tumor by administeringan extended-PK IL-2, and a therapeutic antibody in an amount effectiveto prolong survival in the subject. In one embodiment, the survival ofthe subject, e.g., a mouse model of cancer, is extended by 5 days ormore, 10 days or more, 15 days or more, 20 days or more, 25 days ormore, 30 days or more, 35 days or more, 40 days or more, or 45 days ormore. In another embodiment, the methods of the invention are useful forinhibiting the metastasis of tumors by administering a therapeuticallyeffective amount of extended-PK IL-2 and one or more therapeutic agentsto a subject with established tumors. Preferably, the extended-PK IL-2and therapeutic antibody reduces tumor size to a greater extent thanthat achieved by a combination of IL-2 and one or more therapeuticagents.

In another embodiment, the methods of the invention relate to increasingrecruitment of lymphocytes to the periphery of a tumor in a subject byadministering an extended-PK IL-2 and a therapeutic antibody in anamount effective to increase recruitment of lymphocytes to the peripheryof the tumor.

In yet another embodiment, the methods of the present invention areuseful for stimulating T cell and/or NK cell activity and/orproliferation in a subject by administering an extended-PK IL-2, and atherapeutic antibody in an amount effective to stimulate T cells and/orNK cells in a subject. In another embodiment, the methods of the presentinvention are useful for enhancing antibody-dependent cell-mediatedcytotoxicity (ADCC) and/or cytotoxic T lymphocyte (CTL) responses in asubject comprising administering an extended-PK IL-2, and a therapeuticantibody in an amount effective to enhance ADCC and/or CTL in thesubject. In yet another embodiment, the methods of the invention areuseful for increasing the number of CD8+ T cells in a subject byadministering an extended-PK IL-2, and a therapeutic antibody in anamount effective to increase the number of CD8+ T cells in the subject.In yet another embodiment, the methods of the invention are useful forincreasing the number of NK cells in a subject by administering anextended-PK IL-2, and a therapeutic antibody in an amount effective toincrease the number of NK cells in the subject. In some embodiments thenumber of CD8+ T cells and/or NK cells are increased by 2-fold or more,3-fold or more, 4-fold or more, or 5-fold or more.

In yet another embodiment, the methods of the present invention areuseful for treating cancer and reducing vascular leak syndromeassociated with IL-2 therapy in a subject by administering anextended-PK IL-2, and a therapeutic antibody in an amount effective totreat cancer and reduce vascular leak syndrome associated with IL-2therapy in the subject. In another embodiment, the methods of thepresent invention are useful for treating cancer and reducing pulmonaryedema associated with IL-2 therapy in a subject by administering anextended-pharmacokinetic (PK) interleukin (IL)-2, and a therapeuticantibody in an amount effective to treat cancer and reduce pulmonaryedema associated with IL-2 therapy in the subject.

IL-2 Mutants

Site-directed mutagenesis was used to isolate IL-2 mutants that exhibithigh affinity binding to CD25, i.e., IL-2Rα, as compared to wild-typeIL-2. Increasing the affinity of IL-2 for IL-2Rα at the cell surfacewill increase receptor occupancy within a limited range of IL-2concentration, as well as raise the local concentration of IL-2 at thecell surface.

In one embodiment, the invention features IL-2 mutants, which may be,but are not necessarily, substantially purified and which can functionas high affinity CD25 binders. IL-2 is a T cell growth factor thatinduces proliferation of antigen-activated T cells and stimulation of NKcells. Exemplary IL-2 mutants of the present invention which are highaffinity binders include those shown in FIG. 1, such as those with aminoacid sequences set forth in SEQ ID NOs: 4, 20, 22, 24, 26, and 28.Further exemplary IL-2 mutants with increased affinity for CD25 aredisclosed in U.S. Pat. No. 7,569,215, the contents of which areincorporated herein by reference. In one embodiment, the IL-2 mutant isdoes not bind to CD25, e.g., those with amino acid sequences set forthin SEQ ID NOs: 6 and 8.

IL-2 mutants include an amino acid sequence that is at least 80%identical to SEQ ID NO:30 and that bind CD25. For example, an IL-2mutant can have at least one mutation (e.g., a deletion, addition, orsubstitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, or more amino acid residues) that increases the affinityfor the alpha subunit of the IL-2 receptor relative to wild-type IL-2.It should be understood that mutations identified in mouse IL-2 may bemade at corresponding residues in full length human IL-2 (nucleic acidsequence (accession: NM000586) of SEQ ID NO: 29; amino acid sequence(accession: P60568) of SEQ ID NO: 30) or human IL-2 without the signalpeptide (nucleic acid sequence of SEQ ID NO: 31; amino acid sequence ofSEQ ID NO: 32). Accordingly, in preferred embodiments, the IL-2 moietyof the extended-PK IL-2 is human IL-2. In other embodiments, the IL-2moiety of the extended-PK IL-2 is a mutant human IL-2.

IL-2 mutants can be at least or about 50%, at least or about 65%, atleast or about 70%, at least or about 80%, at least or about 85%, atleast or about 87%, at least or about 90%, at least or about 95%, atleast or about 97%, at least or about 98%, or at least or about 99%identical to wild-type IL-2 (in its precursor form or, preferably, themature form). The mutation can consist of a change in the number orcontent of amino acid residues. For example, the IL-2 mutants can have agreater or a lesser number of amino acid residues than wild-type IL-2.Alternatively, or in addition, IL-2 mutants can contain a substitutionof one or more amino acid residues that are present in the wild-typeIL-2.

By way of illustration, a polypeptide that includes an amino acidsequence that is at least 95% identical to a reference amino acidsequence of SEQ ID NO:30 is a polypeptide that includes a sequence thatis identical to the reference sequence except for the inclusion of up tofive alterations of the reference amino acid of SEQ ID NO:30. Forexample, up to 5% of the amino acid residues in the reference sequencemay be deleted or substituted with another amino acid, or a number ofamino acids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino (N-) or carboxy (C-)terminal positions of the reference amino acid sequence or anywherebetween those terminal positions, interspersed either individually amongresidues in the reference sequence or in one or more contiguous groupswithin the reference sequence.

The substituted amino acid residue(s) can be, but are not necessarily,conservative substitutions, which typically include substitutions withinthe following groups: glycine, alanine; valine, isoleucine, leucine;aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine. These mutations can be atamino acid residues that contact IL-2Rα.

In general, the polypeptides used in the practice of the instantinvention will be synthetic, or produced by expression of a recombinantnucleic acid molecule. In the event the polypeptide is an extended-PKIL-2 (e.g., a fusion protein containing at least IL-2 and a heterologouspolypeptide, such as a hexa-histidine tag or hemagglutinin tag or an Fcregion or human serum albumin), it can be encoded by a hybrid nucleicacid molecule containing one sequence that encodes IL-2 and a secondsequence that encodes all or part of the heterologous polypeptide.

The techniques that are required to make IL-2 mutants are routine in theart, and can be performed without resort to undue experimentation by oneof ordinary skill in the art. For example, a mutation that consists of asubstitution of one or more of the amino acid residues in IL-2 can becreated using a PCR-assisted mutagenesis technique (e.g., as known inthe art and/or described herein for the creation of IL-2 mutants).Mutations that consist of deletions or additions of amino acid residuesto an IL-2 polypeptide can also be made with standard recombinanttechniques. In the event of a deletion or addition, the nucleic acidmolecule encoding IL-2 is simply digested with an appropriaterestriction endonuclease. The resulting fragment can either be expresseddirectly or manipulated further by, for example, ligating it to a secondfragment. The ligation may be facilitated if the two ends of the nucleicacid molecules contain complementary nucleotides that overlap oneanother, but blunt-ended fragments can also be ligated. PCR-generatednucleic acids can also be used to generate various mutant sequences.

In addition to generating IL-2 mutants via expression of nucleic acidmolecules that have been altered by recombinant molecular biologicaltechniques, IL-2 mutants can be chemically synthesized. Chemicallysynthesized polypeptides are routinely generated by those of skill inthe art.

As noted above, IL-2 can also be prepared as fusion or chimericpolypeptides that include IL-2 and a heterologous polypeptide (i.e., apolypeptide that is not IL-2). The heterologous polypeptide can increasethe circulating half-life of the chimeric polypeptide in vivo, and may,therefore, further enhance the properties of IL-2. As discussed infurther detail infra, the polypeptide that increases the circulatinghalf-life may be a serum albumin, such as human serum albumin, or the Fcregion of the IgG subclass of antibodies that lacks the IgG heavy chainvariable region. The Fc region can include a mutation that inhibitseffector functions such as complement fixation and Fc receptor binding.

In other embodiments, the chimeric polypeptide can include IL-2 and apolypeptide that functions as an antigenic tag, such as a FLAG sequence.FLAG sequences are recognized by biotinylated, highly specific,anti-FLAG antibodies, as described herein (see also Blanar et al.,Science 256:1014, 1992; LeClair et al., Proc. Natl. Acad. Sci. USA89:8145, 1992). In some embodiments, the chimeric polypeptide furthercomprises a C-terminal c-myc epitope tag.

Chimeric polypeptides can be constructed using no more than conventionalmolecular biological techniques, which are well within the ability ofthose of ordinary skill in the art to perform.

Nucleic Acid Molecules Encoding IL-2 Mutants

IL-2, either alone or as a part of a chimeric polypeptide, such as thosedescribed above, can be obtained by expression of a nucleic acidmolecule. Thus, nucleic acid molecules encoding polypeptides containingan IL-2 mutant are considered within the scope of the invention, such asthose with nucleic acid sequences set forth in SEQ ID NOs: 1, 3, 5, 7,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31. Just as IL-2 mutants canbe described in terms of their identity with wild-type IL-2, the nucleicacid molecules encoding them will necessarily have a certain identitywith those that encode wild-type IL-2. For example, the nucleic acidmolecule encoding an IL-2 mutant can be at least 50%, at least 65%,preferably at least 75%, more preferably at least 85%, and mostpreferably at least 95% (e.g., 99%) identical to the nucleic acidencoding full length wild-type IL-2 (e.g., SEQ ID NO:29) or wild-typeIL-2 without the signal peptide (e.g., SEQ ID NO: 31).

The nucleic acid molecules of the invention can contain naturallyoccurring sequences, or sequences that differ from those that occurnaturally, but, due to the degeneracy of the genetic code, encode thesame polypeptide. These nucleic acid molecules can consist of RNA or DNA(for example, genomic DNA, cDNA, or synthetic DNA, such as that producedby phosphoramidite-based synthesis), or combinations or modifications ofthe nucleotides within these types of nucleic acids. In addition, thenucleic acid molecules can be double-stranded or single-stranded (i.e.,either a sense or an antisense strand).

The nucleic acid molecules are not limited to sequences that encodepolypeptides; some or all of the non-coding sequences that lie upstreamor downstream from a coding sequence (e.g., the coding sequence of IL-2)can also be included. Those of ordinary skill in the art of molecularbiology are familiar with routine procedures for isolating nucleic acidmolecules. They can, for example, be generated by treatment of genomicDNA with restriction endonucleases, or by performance of the polymerasechain reaction (PCR). In the event the nucleic acid molecule is aribonucleic acid (RNA), molecules can be produced, for example, by invitro transcription.

The isolated nucleic acid molecules of the invention can includefragments not found as such in the natural state. Thus, the inventionencompasses recombinant molecules, such as those in which a nucleic acidsequence (for example, a sequence encoding an IL-2 mutant) isincorporated into a vector (e.g., a plasmid or viral vector) or into thegenome of a heterologous cell (or the genome of a homologous cell, at aposition other than the natural chromosomal location).

As described above, IL-2 mutants of the invention may exist as a part ofa chimeric polypeptide. In addition to, or in place of, the heterologouspolypeptides described above, a nucleic acid molecule of the inventioncan contain sequences encoding a “marker” or “reporter.” Examples ofmarker or reporter genes include .beta.-lactamase, chloramphenicolacetyltransferase (CAT), adenosine deaminase (ADA), aminoglycosidephosphotransferase (neo^(r), G418^(r)), dihydrofolate reductase (DHFR),hygromycin-B-hosphotransferase (HPH), thymidine kinase (TK), lacz(encoding β-galactosidase), and xanthine guaninephosphoribosyltransferase (XGPRT). As with many of the standardprocedures associated with the practice of the invention, skilledartisans will be aware of additional useful reagents, for example, ofadditional sequences that can serve the function of a marker orreporter.

The nucleic acid molecules of the invention can be obtained byintroducing a mutation into IL-2-encoding DNA obtained from anybiological cell, such as the cell of a mammal. Thus, the nucleic acidsof the invention (and the polypeptides they encode) can be those of amouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, monkey, baboon,dog, or cat. Typically, the nucleic acid molecules will be those of ahuman.

Expression of IL-2 Mutants

The nucleic acid molecules described above can be contained within avector that is capable of directing their expression in, for example, acell that has been transduced with the vector. Accordingly, in additionto IL-2 mutants, expression vectors containing a nucleic acid moleculeencoding an IL-2 mutant and cells transfected with these vectors areamong the preferred embodiments.

Vectors suitable for use in the present invention include T7-basedvectors for use in bacteria (see, for example, Rosenberg et al., Gene56:125, 1987), the pMSXND expression vector for use in mammalian cells(Lee and Nathans, J. Biol. Chem. 263:3521, 1988), andbaculovirus-derived vectors (for example the expression vector pBacPAK9from Clontech, Palo Alto, Calif.) for use in insect cells. The nucleicacid inserts, which encode the polypeptide of interest in such vectors,can be operably linked to a promoter, which is selected based on, forexample, the cell type in which expression is sought. For example, a T7promoter can be used in bacteria, a polyhedrin promoter can be used ininsect cells, and a cytomegalovirus or metallothionein promoter can beused in mammalian cells. Also, in the case of higher eukaryotes,tissue-specific and cell type-specific promoters are widely available.These promoters are so named for their ability to direct expression of anucleic acid molecule in a given tissue or cell type within the body.Skilled artisans are well aware of numerous promoters and otherregulatory elements which can be used to direct expression of nucleicacids.

In addition to sequences that facilitate transcription of the insertednucleic acid molecule, vectors can contain origins of replication, andother genes that encode a selectable marker. For example, theneomycin-resistance (neo^(r)) gene imparts G418 resistance to cells inwhich it is expressed, and thus permits phenotypic selection of thetransfected cells. Those of skill in the art can readily determinewhether a given regulatory element or selectable marker is suitable foruse in a particular experimental context.

Viral vectors that can be used in the invention include, for example,retroviral, adenoviral, and adeno-associated vectors, herpes virus,simian virus 40 (SV40), and bovine papilloma virus vectors (see, forexample, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press,Cold Spring Harbor, N.Y.).

Prokaryotic or eukaryotic cells that contain and express a nucleic acidmolecule that encodes an IL-2 mutant are also features of the invention.A cell of the invention is a transfected cell, i.e., a cell into which anucleic acid molecule, for example a nucleic acid molecule encoding anIL-2 mutant, has been introduced by means of recombinant DNA techniques.The progeny of such a cell are also considered within the scope of theinvention.

The precise components of the expression system are not critical. Forexample, an IL-2 mutant can be produced in a prokaryotic host, such asthe bacterium E. coli, or in a eukaryotic host, such as an insect cell(e.g., an Sf21 cell), or mammalian cells (e.g., COS cells, NIH 3T3cells, or HeLa cells). These cells are available from many sources,including the American Type Culture Collection (Manassas, Va.). Inselecting an expression system, it matters only that the components arecompatible with one another. Artisans or ordinary skill are able to makesuch a determination. Furthermore, if guidance is required in selectingan expression system, skilled artisans may consult Ausubel et al.(Current Protocols in Molecular Biology, John Wiley and Sons, New York,N.Y., 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual,1985 Suppl. 1987).

The expressed polypeptides can be purified from the expression systemusing routine biochemical procedures, and can be used, e.g., astherapeutic agents, as described herein.

Extended-PK Groups

As described supra, IL-2 or mutant IL-2 is fused to an extended-PKgroup, which increases circulation half-life. Non-limiting examples ofextended-PK groups are described infra. It should be understood thatother PK groups that increase the circulation half-life of IL-2, orvariants thereof, are also applicable to the present invention. In apreferred embodiment, the extended-PK group is a Fc domain.

In some embodiments, the serum half-life of extended-PK IL-2 isincreased relative to IL-2 alone (i.e., IL-2 not fused to an extended-PKgroup). In certain embodiments, the serum half-life of extended-PK IL-2is at least 20, 40, 60, 80, 100, 120, 150, 180, 200, 400, 600, 800, or1000% longer relative to the serum half-life of IL-2 alone. In otherembodiments, the serum half-life of the extended-PK IL-2 is at least1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5 fold, 4-fold, 4.5-fold, 5-fold,6-fold, 7-fold, 8-fold, 10-fold, 12-fold, 13-fold, 15-fold, 17-fold,20-fold, 22-fold, 25-fold, 27-fold, 30-fold, 35-fold, 40-fold, or50-fold greater than the serum half-life of IL-2 alone. In someembodiments, the serum half-life of the extended-PK IL-2 is at least 10hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200hours.

Fc Domains

In some embodiments, an extended-PK IL-2 includes an Fc domain, such asthat with an amino acid sequences set forth in SEQ ID NO: 34. It will beunderstood by those in the art that epitope tags corresponding to 6× histag on these extended-PK IL-2 with Fc domains are optional. The Fcdomain does not contain a variable region that binds to antigen. Fcdomains useful for producing the extended-PK IL-2 of the presentinvention may be obtained from a number of different sources. Inpreferred embodiments, an Fc domain of the extended-PK IL-2 is derivedfrom a human immunoglobulin. In a preferred embodiment, the Fc domain isfrom a human IgG1 constant region (SEQ ID NO: 33). The Fc domain ofhuman IgG1 is set forth in SEQ ID NO: 34. It is understood, however,that the Fc domain may be derived from an immunoglobulin of anothermammalian species, including for example, a rodent (e.g. a mouse, rat,rabbit, guinea pig) or non-human primate (e.g. chimpanzee, macaque)species. Moreover, the Fc domain or portion thereof may be derived fromany immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and anyimmunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4.

In some aspects, an extended-PK IL-2 includes a mutant Fc domain. Insome aspects, an extended-PK IL-2 includes a mutant, IgG1 Fc domain. Insome aspects, a mutant Fc domain comprises one or more mutations in thehinge, CH2, and/or CH3 domains. In some aspects, a mutant Fc domainincludes a D265A mutation.

A variety of Fc domain gene sequences (e.g., mouse and human constantregion gene sequences) are available in the form of publicly accessibledeposits. Constant region domains comprising an Fc domain sequence canbe selected lacking a particular effector function and/or with aparticular modification to reduce immunogenicity. Many sequences ofantibodies and antibody-encoding genes have been published and suitableFc domain sequences (e.g. hinge, CH2, and/or CH3 sequences, or portionsthereof) can be derived from these sequences using art recognizedtechniques. The genetic material obtained using any of the foregoingmethods may then be altered or synthesized to obtain polypeptides of thepresent invention. It will further be appreciated that the scope of thisinvention encompasses alleles, variants and mutations of constant regionDNA sequences.

Fc domain sequences can be cloned, e.g., using the polymerase chainreaction and primers which are selected to amplify the domain ofinterest. To clone an Fc domain sequence from an antibody, mRNA can beisolated from hybridoma, spleen, or lymph cells, reverse transcribedinto DNA, and antibody genes amplified by PCR. PCR amplification methodsare described in detail in U.S. Pat. Nos. 4,683,195; 4,683,202;4,800,159; 4,965,188; and in, e.g., “PCR Protocols: A Guide to Methodsand Applications” Innis et al. eds., Academic Press, San Diego, Calif.(1990); Ho et al. 1989. Gene 77:51; Horton et al. 1993. Methods Enzymol.217:270). PCR may be initiated by consensus constant region primers orby more specific primers based on the published heavy and light chainDNA and amino acid sequences. As discussed above, PCR also may be usedto isolate DNA clones encoding the antibody light and heavy chains. Inthis case the libraries may be screened by consensus primers or largerhomologous probes, such as mouse constant region probes. Numerous primersets suitable for amplification of antibody genes are known in the art(e.g., 5′ primers based on the N-terminal sequence of purifiedantibodies (Benhar and Pastan. 1994. Protein Engineering 7:1509); rapidamplification of cDNA ends (Ruberti, F. et al. 1994. J. Immunol. Methods173:33); antibody leader sequences (Larrick et al. Biochem Biophys ResCommun 1989; 160:1250). The cloning of antibody sequences is furtherdescribed in Newman et al., U.S. Pat. No. 5,658,570, filed Jan. 25,1995, which is herein incorporated by reference.

Extended-PK IL-2 of the invention may comprise one or more Fc domains(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Fc domains). In oneembodiment, the Fc domains may be of different types. In one embodiment,at least one Fc domain present in the extended-PK IL-2 comprises a hingedomain or portion thereof. In another embodiment, the extended-PK IL-2of the invention comprises at least one Fc domain which comprises atleast one CH2 domain or portion thereof. In another embodiment, theextended-PK IL-2 of the invention comprises at least one Fc domain whichcomprises at least one CH3 domain or portion thereof. In anotherembodiment, the extended-PK IL-2 of the invention comprises at least oneFc domain which comprises at least one CH4 domain or portion thereof. Inanother embodiment, the extended-PK IL-2 of the invention comprises atleast one Fc domain which comprises at least one hinge domain or portionthereof and at least one CH2 domain or portion thereof (e.g, in thehinge-CH2 orientation). In another embodiment, the extended-PK IL-2 ofthe invention comprises at least one Fc domain which comprises at leastone CH2 domain or portion thereof and at least one CH3 domain or portionthereof (e.g, in the CH2-CH3 orientation). In another embodiment, theextended-PK IL-2 of the invention comprises at least one Fc domaincomprising at least one hinge domain or portion thereof, at least oneCH2 domain or portion thereof, and least one CH3 domain or portionthereof, for example in the orientation hinge-CH2-CH3, hinge-CH3-CH2, orCH2-CH3-hinge.

In certain embodiments, extended-PK IL-2 comprises at least one completeFc region derived from one or more immunoglobulin heavy chains (e.g., anFc domain including hinge, CH2, and CH3 domains, although these need notbe derived from the same antibody). In other embodiments, extended-PKIL-2 comprises at least two complete Fc domains derived from one or moreimmunoglobulin heavy chains. In preferred embodiments, the complete Fcdomain is derived from a human IgG immunoglobulin heavy chain (e.g.,human IgG1).

In another embodiment, the extended-PK IL-2 of the invention comprisesat least one Fc domain comprising a complete CH3 domain. In anotherembodiment, the extended-PK IL-2 of the invention comprises at least oneFc domain comprising a complete CH2 domain. In another embodiment, theextended-PK IL-2 of the invention comprises at least one Fc domaincomprising at least a CH3 domain, and at least one of a hinge region,and a CH2 domain. In one embodiment, the extended-PK IL-2 of theinvention comprises at least one Fc domain comprising a hinge and a CH3domain. In another embodiment, the extended-PK IL-2 of the inventioncomprises at least one Fc domain comprising a hinge, a CH2, and a CH3domain. In preferred embodiments, the Fc domain is derived from a humanIgG immunoglobulin heavy chain (e.g., human IgG1).

The constant region domains or portions thereof making up an Fc domainof the extended-PK IL-2 of the invention may be derived from differentimmunoglobulin molecules. For example, a polypeptide of the inventionmay comprise a CH2 domain or portion thereof derived from an IgG1molecule and a CH3 region or portion thereof derived from an IgG3molecule. In another example, the extended-PK IL-2 can comprise an Fcdomain comprising a hinge domain derived, in part, from an IgG1 moleculeand, in part, from an IgG3 molecule. As set forth herein, it will beunderstood by one of ordinary skill in the art that an Fc domain may bealtered such that it varies in amino acid sequence from a naturallyoccurring antibody molecule.

In one embodiment, the extended-PK IL-2 of the invention lacks one ormore constant region domains of a complete Fc region, i.e., they arepartially or entirely deleted. In certain embodiments, the extended-PKIL-2 of the invention will lack an entire CH2 domain. In certainembodiments, the extended-PK IL-2 of the invention comprise CH2domain-deleted Fc regions derived from a vector (e.g., from IDECPharmaceuticals, San Diego) encoding an IgG1 human constant regiondomain (see, e.g., WO02/060955A2 and WO02/096948A2). This exemplaryvector is engineered to delete the CH2 domain and provide a syntheticvector expressing a domain-deleted IgG1 constant region. It will benoted that these exemplary constructs are preferably engineered to fusea binding CH3 domain directly to a hinge region of the respective Fcdomain.

In other constructs it may be desirable to provide a peptide spacerbetween one or more constituent Fc domains. For example, a peptidespacer may be placed between a hinge region and a CH2 domain and/orbetween a CH2 and a CH3 domain. For example, compatible constructs couldbe expressed wherein the CH2 domain has been deleted and the remainingCH3 domain (synthetic or unsynthetic) is joined to the hinge region witha 1-20, 1-10, or 1-5 amino acid peptide spacer. Such a peptide spacermay be added, for instance, to ensure that the regulatory elements ofthe constant region domain remain free and accessible or that the hingeregion remains flexible. Preferably, any linker peptide compatible withthe instant invention will be relatively non-immunogenic and not preventproper folding of the Fc.

Changes to Fc Amino Acids

In certain embodiments, an Fc domain employed in the extended-PK IL-2 ofthe invention is altered or modified, e.g., by amino acid mutation(e.g., addition, deletion, or substitution). As used herein, the term“Fc domain variant” refers to an Fc domain having at least one aminoacid modification, such as an amino acid substitution, as compared tothe wild-type Fc from which the Fc domain is derived. For example,wherein the Fc domain is derived from a human IgG1 antibody, a variantcomprises at least one amino acid mutation (e.g., substitution) ascompared to a wild type amino acid at the corresponding position of thehuman IgG1 Fc region.

In one embodiment, the Fc variant comprises a substitution at an aminoacid position located in a hinge domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH2 domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH3 domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH4 domain or portion thereof.

In certain embodiments, the extended-PK IL-2 of the invention comprisean Fc variant comprising more than one amino acid substitution. Theextended-PK IL-2 of the invention may comprise, for example, 2, 3, 4, 5,6, 7, 8, 9, 10 or more amino acid substitutions. Preferably, the aminoacid substitutions are spatially positioned from each other by aninterval of at least 1 amino acid position or more, for example, atleast 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid positions or more. Morepreferably, the engineered amino acids are spatially positioned apartfrom each other by an interval of at least 5, 10, 15, 20, or 25 aminoacid positions or more.

In some aspects, an Fc domain includes changes in the region betweenamino acids 234-238, including the sequence LLGGP at the beginning ofthe CH2 domain. In some aspects, an Fc variant alters Fc mediatedeffector function, particularly ADCC, and/or decrease binding avidityfor Fc receptors. In some aspects, sequence changes closer to theCH2-CH3 junction, at positions such as K322 or P331 can eliminatecomplement mediated cytotoxicity and/or alter avidity for FcR binding.In some aspects, an Fc domain incorporates changes at residues P238 andP331, e.g., changing the wild type prolines at these positions toserine. In some aspects, alterations in the hinge region at one or moreof the three hinge cysteines, to encode CCC, SCC, SSC, SCS, or SSS atthese residues can also affect FcR binding and molecular homogeneity,e.g., by elimination of unpaired cysteines that may destabilize thefolded protein.

Other amino acid mutations in the Fc domain are contemplated to reducebinding to the Fc gamma receptor and Fc gamma receptor subtypes. Forexample, mutations at positions 238, 239, 248, 249, 252, 254, 255, 256,258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290,292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 322, 324,327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 356, 360, 373, 376,378, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or439 of the Fc region can alter binding as described in U.S. Pat. No.6,737,056, issued May 18, 2004, incorporated herein by reference in itsentirety. This patent reported that changing Pro331 in IgG3 to Serresulted in six fold lower affinity as compared to unmutated IgG3,indicating the involvement of Pro331 in Fc gamma RI binding. Inaddition, amino acid modifications at positions 234, 235, 236, and 237,297, 318, 320 and 322 are disclosed as potentially altering receptorbinding affinity in U.S. Pat. No. 5,624,821, issued Apr. 29, 1997 andincorporated herein by reference in its entirety.

Further mutations contemplated for use include, e.g., those described inU.S. Pat. App. Pub. No. 2006/0235208, published Oct. 19, 2006 andincorporated herein by reference in its entirety. This publicationdescribes Fc variants that exhibit reduced binding to Fc gammareceptors, reduced antibody dependent cell-mediated cytotoxicity, orreduced complement dependent cytotoxicity, that comprise at least oneamino acid modification in the Fc region, including 232G, 234G, 234H,235D, 235G, 235H, 236I, 236N, 236P, 236R, 237K, 237L, 237N, 237P, 238K,239R, 265G, 267R, 269R, 270H, 297S, 299A, 299I, 299V, 325A, 325L, 327R,328R, 329K, 330I, 330L, 330N, 330P, 330R, and 331L (numbering isaccording to the EU index), as well as double mutants 236R/237K,236R/325L, 236R/328R, 237K/325L, 237K/328R, 325L/328R, 235G/236R,267R/269R, 234G/235G, 236R/237K/325L, 236R/325L/328R, 235G/236R/237K,and 237K/325L/328R. Other mutations contemplated for use as described inthis publication include 227G, 234D, 234E, 234G, 234I, 234Y, 235D, 2351,235S, 236S, 239D, 246H, 255Y, 258H, 260H, 2641, 267D, 267E, 268D, 268E,272H, 272I, 272R, 281D, 282G, 283H, 284E, 293R, 295E, 304T, 324G, 324I,327D, 327A, 328A, 328D, 328E, 328F, 3281, 328M, 328N, 328Q, 328T, 328V,328Y, 330I, 330L, 330Y, 332D, 332E, 335D, an insertion of G betweenpositions 235 and 236, an insertion of A between positions 235 and 236,an insertion of S between positions 235 and 236, an insertion of Tbetween positions 235 and 236, an insertion of N between positions 235and 236, an insertion of D between positions 235 and 236, an insertionof V between positions 235 and 236, an insertion of L between positions235 and 236, an insertion of G between positions 235 and 236, aninsertion of A between positions 235 and 236, an insertion of S betweenpositions 235 and 236, an insertion of T between positions 235 and 236,an insertion of N between positions 235 and 236, an insertion of Dbetween positions 235 and 236, an insertion of V between positions 235and 236, an insertion of L between positions 235 and 236, an insertionof G between positions 297 and 298, an insertion of A between positions297 and 298, an insertion of S between positions 297 and 298, aninsertion of D between positions 297 and 298, an insertion of G betweenpositions 326 and 327, an insertion of A between positions 326 and 327,an insertion of T between positions 326 and 327, an insertion of Dbetween positions 326 and 327, and an insertion of E between positions326 and 327 (numbering is according to the EU index). Additionally,mutations described in U.S. Pat. App. Pub. No. 2006/0235208 include227G/332E, 234D/332E, 234E/332E, 234Y/332E, 234I/332E, 234G/332E,235I/332E, 235S/332E, 235D/332E, 235E/332E, 236S/332E, 236A/332E,236S/332D, 236A/332D, 239D/268E, 246H/332E, 255Y/332E, 258H/332E,260H/332E, 264I/332E, 267E/332E, 267D/332E, 268D/332D, 268E/332D,268E/332E, 268D/332E, 268E/330Y, 268D/330Y, 272R/332E, 272H/332E,283H/332E, 284E/332E, 293R/332E, 295E/332E, 304T/332E, 324I/332E,324G/332E, 324I/332D, 324G/332D, 327D/332E, 328A/332E, 328T/332E,328V/332E, 328I/332E, 328F/332E, 328Y/332E, 328M/332E, 328D/332E,328E/332E, 328N/332E, 328Q/332E, 328A/332D, 328T/332D, 328V/332D,328I/332D, 328F/332D, 328Y/332D, 328M/332D, 328D/332D, 328E/332D,328N/332D, 328Q/332D, 330L/332E, 330Y/332E, 330I/332E, 332D/330Y,335D/332E, 239D/332E, 239D/332E/330Y, 239D/332E/330L, 239D/332E/330I,239D/332E/268E, 239D/332E/268D, 239D/332E/327D, 239D/332E/284E,239D/268E/330Y, 239D/332E/268E/330Y, 239D/332E/327A,239D/332E/268E/327A, 239D/332E/330Y/327A, 332E/330Y/268 E/327A,239D/332E/268E/330Y/327A, Insert G>297-298/332E, Insert A>297-298/332E,Insert S>297-298/332E, Insert D>297-298/332E, Insert G>326-327/332E,Insert A>326-327/332E, Insert T>326-327/332E, Insert D>326-327/332E,Insert E>326-327/332E, Insert G>235-236/332E, Insert A>235-236/332E,Insert S>235-236/332E, Insert T>235-236/332E, Insert N>235-236/332E,Insert D>235-236/332E, Insert V>235-236/332E, Insert L>235-236/332E,Insert G>235-236/332D, Insert A>235-236/332D, Insert S>235-236/332D,Insert T>235-236/332D, Insert N>235-236/332D, Insert D>235-236/332D,Insert V>235-236/332D, and Insert L>235-236/332D (numbering according tothe EU index) are contemplated for use. The mutant L234A/L235A isdescribed, e.g., in U.S. Pat. App. Pub. No. 2003/0108548, published Jun.12, 2003 and incorporated herein by reference in its entirety. Inembodiments, the described modifications are included eitherindividually or in combination. In a preferred embodiment, the mutationis D265A in human IgG 1.

In certain embodiments, the extended-PK IL-2 of the invention comprisesan amino acid substitution to an Fc domain which alters theantigen-independent effector functions of the antibody, in particularthe circulating half-life of the antibody.

In other embodiments, the extended-PK IL-2 of the invention comprises anFc variant comprising an amino acid substitution which alters theantigen-dependent effector functions of the polypeptide, in particularADCC or complement activation, e.g., as compared to a wild type Fcregion. Such extended-PK IL-2 exhibit decreased binding to FcR gammawhen compared to wild-type polypeptides and, therefore, mediate reducedeffector function. Fc variants with decreased FcR gamma binding affinityare expected to reduce effector function, and such molecules are alsouseful, for example, for treatment of conditions in which target celldestruction is undesirable, e.g., where normal cells may express targetmolecules, or where chronic administration of the polypeptide mightresult in unwanted immune system activation.

In one embodiment, the extended-PK IL-2 exhibits altered binding to anactivating FcγR (e.g. FcγI, FcγIIa, or FcγRIIIa). In another embodiment,the extended-PK IL-2 exhibits altered binding affinity to an inhibitoryFcγR (e.g. FcγRIIb). Exemplary amino acid substitutions which alteredFcR or complement binding activity are disclosed in International PCTPublication No. WO05/063815 which is incorporated by reference herein.

The extended-PK IL-2 of the invention may also comprise an amino acidsubstitution which alters the glycosylation of the extended-PK IL-2. Forexample, the Fc domain of the extended-PK IL-2 may comprise an Fc domainhaving a mutation leading to reduced glycosylation (e.g., N- or O-linkedglycosylation) or may comprise an altered glycoform of the wild-type Fcdomain (e.g., a low fucose or fucose-free glycan). In anotherembodiment, the extended-PK IL-2 has an amino acid substitution near orwithin a glycosylation motif, for example, an N-linked glycosylationmotif that contains the amino acid sequence NXT or NXS. Exemplary aminoacid substitutions which reduce or alter glycosylation are disclosed inWO05/018572 and US2007/0111281, which are incorporated by referenceherein.

In other embodiments, the extended-PK IL-2 of the invention comprises atleast one Fc domain having engineered cysteine residue or analog thereofwhich is located at the solvent-exposed surface. In preferredembodiments, the extended-PK IL-2 of the invention comprise an Fc domaincomprising at least one engineered free cysteine residue or analogthereof that is substantially free of disulfide bonding with a secondcysteine residue. Any of the above engineered cysteine residues oranalogs thereof may subsequently be conjugated to a functional domainusing art-recognized techniques (e.g., conjugated with a thiol-reactiveheterobifunctional linker).

In one embodiment, the extended-PK IL-2 of the invention may comprise agenetically fused Fc domain having two or more of its constituent Fcdomains independently selected from the Fc domains described herein. Inone embodiment, the Fc domains are the same. In another embodiment, atleast two of the Fc domains are different. For example, the Fc domainsof the extended-PK IL-2 of the invention comprise the same number ofamino acid residues or they may differ in length by one or more aminoacid residues (e.g., by about 5 amino acid residues (e.g., 1, 2, 3, 4,or 5 amino acid residues), about 10 residues, about 15 residues, about20 residues, about 30 residues, about 40 residues, or about 50residues). In yet other embodiments, the Fc domains of the extended-PKIL-2 of the invention may differ in sequence at one or more amino acidpositions. For example, at least two of the Fc domains may differ atabout 5 amino acid positions (e.g., 1, 2, 3, 4, or 5 amino acidpositions), about 10 positions, about 15 positions, about 20 positions,about 30 positions, about 40 positions, or about 50 positions).

PEGylation

In some embodiments, an extended-PK IL-2 of the present inventionincludes a polyethylene glycol (PEG) domain. PEGylation is well known inthe art to confer increased circulation half-life to proteins. Methodsof PEGylation are well known and disclosed in, e.g., U.S. Pat. No.7,610,156, U.S. Pat. No. 7,847,062, all of which are hereby incorporatedby reference.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). Theterm “PEG” is used broadly to encompass any polyethylene glycolmolecule, without regard to size or to modification at an end of thePEG, and can be represented by the formula: X—O(CH₂CH₂O)_(n-1)CH₂CH₂OH,where n is 20 to 2300 and X is H or a terminal modification, e.g., aC₁₋₄ alkyl. In one embodiment, the PEG of the invention terminates onone end with hydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”).PEG can contain further chemical groups which are necessary for bindingreactions; which results from the chemical synthesis of the molecule; orwhich is a spacer for optimal distance of parts of the molecule. Inaddition, such a PEG can consist of one or more PEG side-chains whichare linked together. PEGs with more than one PEG chain are calledmultiarmed or branched PEGs. Branched PEGs can be prepared, for example,by the addition of polyethylene oxide to various polyols, includingglycerol, pentaerythriol, and sorbitol. For example, a four-armedbranched PEG can be prepared from pentaerythriol and ethylene oxide.Branched PEG are described in, for example, EP-A 0 473 084 and U.S. Pat.No. 5,932,462, both of which are hereby incorporated by reference. Oneform of PEGs includes two PEG side-chains (PEG2) linked via the primaryamino groups of a lysine (Monfardini et al., Bioconjugate Chem 1995;6:62-9).

In one embodiment, pegylated IL-2 is produced by site-directedpegylation, particularly by conjugation of PEG to a cysteine moiety atthe N- or C-terminus. A PEG moiety may also be attached by otherchemistry, including by conjugation to amines.

PEG conjugation to peptides or proteins generally involves theactivation of PEG and coupling of the activated PEG-intermediatesdirectly to target proteins/peptides or to a linker, which issubsequently activated and coupled to target proteins/peptides (seeAbuchowski et al., JBC 1977; 252:3571 and JBC 1977; 252:3582, and Harriset. al., in: Poly(ethylene glycol) Chemistry: Biotechnical andBiomedical Applications; (J. M. Harris ed.) Plenum Press: New York,1992; Chap. 21 and 22).

A variety of molecular mass forms of PEG can be selected, e.g., fromabout 1,000 Daltons (Da) to 100,000 Da (n is 20 to 2300), forconjugating to IL-2. The number of repeating units “n” in the PEG isapproximated for the molecular mass described in Daltons. It ispreferred that the combined molecular mass of PEG on an activated linkeris suitable for pharmaceutical use. Thus, in one embodiment, themolecular mass of the PEG molecules does not exceed 100,000 Da. Forexample, if three PEG molecules are attached to a linker, where each PEGmolecule has the same molecular mass of 12,000 Da (each n is about 270),then the total molecular mass of PEG on the linker is about 36,000 Da(total n is about 820). The molecular masses of the PEG attached to thelinker can also be different, e.g., of three molecules on a linker twoPEG molecules can be 5,000 Da each (each n is about 110) and one PEGmolecule can be 12,000 Da (n is about 270).

One skilled in the art can select a suitable molecular mass for PEG,e.g., based on how the pegylated IL-2 will be used therapeutically, thedesired dosage, circulation time, resistance to proteolysis,immunogenicity, and other considerations. For a discussion of PEG andits use to enhance the properties of proteins, see N. V. Katre, AdvancedDrug Delivery Reviews 1993; 10:91-114.

In one embodiment of the invention, PEG molecules may be activated toreact with amino groups on IL-2 such as with lysines (Bencham C. O. etal., Anal. Biochem., 131, 25 (1983); Veronese, F. M. et al., Appl.Biochem., 11, 141 (1985); Zalipsky, S. et al., Polymeric Drugs and DrugDelivery Systems, adrs 9-110 ACS Symposium Series 469 (1999); Zalipsky,S. et al., Europ. Polym. J., 19, 1177-1183 (1983); Delgado, C. et al.,Biotechnology and Applied Biochemistry, 12, 119-128 (1990)).

In one embodiment, carbonate esters of PEG are used to form the PEG-IL-2conjugates. N,N′-disuccinimidylcarbonate (DSC) may be used in thereaction with PEG to form active mixed PEG-succinimidyl carbonate thatmay be subsequently reacted with a nucleophilic group of a linker or anamino group of IL-2 (see U.S. Pat. No. 5,281,698 and U.S. Pat. No.5,932,462). In a similar type of reaction,1,1′-(dibenzotriazolyl)carbonate and di-(2-pyridyl)carbonate may bereacted with PEG to form PEG-benzotriazolyl and PEG-pyridyl mixedcarbonate (U.S. Pat. No. 5,382,657), respectively.

Pegylation of IL-2 can be performed according to the methods of thestate of the art, for example by reaction of IL-2 with electrophilicallyactive PEGs (Shearwater Corp., USA, www.shearwatercorp.com). PreferredPEG reagents of the present invention are, e.g., N-hydroxysuccinimidylpropionates (PEG-SPA), butanoates (PEG-SBA), PEG-succinimidyl propionateor branched N-hydroxysuccinimides such as mPEG2-NHS (Monfardini, C., etal., Bioconjugate Chem. 6 (1995) 62-69).

In another embodiment, PEG molecules may be coupled to sulfhydryl groupson IL-2 (Sartore, L., et al., Appl. Biochem. Biotechnol., 27, 45 (1991);Morpurgo et al., Biocon. Chem., 7, 363-368 (1996); Goodson et al.,Bio/Technology (1990) 8, 343; U.S. Pat. No. 5,766,897). U.S. Pat. No.6,610,281 and U.S. Pat. No. 5,766,897 describe exemplary reactive PEGspecies that may be coupled to sulfhydryl groups.

In some embodiments where PEG molecules are conjugated to cysteineresidues on IL-2 the cysteine residues are native to IL-2 whereas inother embodiments, one or more cysteine residues are engineered intoIL-2. Mutations may be introduced into the coding sequence of IL-2 togenerate cysteine residues. This might be achieved, for example, bymutating one or more amino acid residues to cysteine. Preferred aminoacids for mutating to a cysteine residue include serine, threonine,alanine and other hydrophilic residues. Preferably, the residue to bemutated to cysteine is a surface-exposed residue. Algorithms arewell-known in the art for predicting surface accessibility of residuesbased on primary sequence or a protein.

In another embodiment, pegylated IL-2 comprise one or more PEG moleculescovalently attached to a linker.

In one embodiment, IL-2 is pegylated at the C-terminus. In a specificembodiment, a protein is pegylated at the C-terminus by the introductionof C-terminal azido-methionine and the subsequent conjugation of amethyl-PEG-triarylphosphine compound via the Staudinger reaction. ThisC-terminal conjugation method is described in Cazalis et al., C-TerminalSite-Specific PEGylation of a Truncated Thrombomodulin Mutant withRetention of Full Bioactivity, Bioconjug Chem. 2004; 15(5):1005-1009.

Monopegylation of IL-2 can also be achieved according to the generalmethods described in WO 94/01451. WO 94/01451 describes a method forpreparing a recombinant polypeptide with a modified terminal amino acidalpha-carbon reactive group. The steps of the method involve forming therecombinant polypeptide and protecting it with one or more biologicallyadded protecting groups at the N-terminal alpha-amine and C-terminalalpha-carboxyl. The polypeptide can then be reacted with chemicalprotecting agents to selectively protect reactive side chain groups andthereby prevent side chain groups from being modified. The polypeptideis then cleaved with a cleavage reagent specific for the biologicalprotecting group to form an unprotected terminal amino acid alpha-carbonreactive group. The unprotected terminal amino acid alpha-carbonreactive group is modified with a chemical modifying agent. The sidechain protected terminally modified single copy polypeptide is thendeprotected at the side chain groups to form a terminally modifiedrecombinant single copy polypeptide. The number and sequence of steps inthe method can be varied to achieve selective modification at the N-and/or C-terminal amino acid of the polypeptide.

The ratio of IL-2 to activated PEG in the conjugation reaction can befrom about 1:0.5 to 1:50, between from about 1:1 to 1:30, or from about1:5 to 1:15. Various aqueous buffers can be used to catalyze thecovalent addition of PEG to IL-2, or variants thereof. In oneembodiment, the pH of a buffer used is from about 7.0 to 9.0. In anotherembodiment, the pH is in a slightly basic range, e.g., from about 7.5 to8.5. Buffers having a pKa close to neutral pH range may be used, e.g.,phosphate buffer.

Conventional separation and purification techniques known in the art canbe used to purify PEGylated IL-2, such as size exclusion (e.g. gelfiltration) and ion exchange chromatography. Products may also beseparated using SDS-PAGE. Products that may be separated include mono-,di-, tri-poly- and un-pegylated IL-2 as well as free PEG. The percentageof mono-PEG conjugates can be controlled by pooling broader fractionsaround the elution peak to increase the percentage of mono-PEG in thecomposition.

In one embodiment, PEGylated IL-2 of the invention contain one, two ormore PEG moieties. In one embodiment, the PEG moiety(ies) are bound toan amino acid residue which is on the surface of the protein and/or awayfrom the surface that contacts CD25. In one embodiment, the combined ortotal molecular mass of PEG in PEG-IL-2 is from about 3,000 Da to 60,000Da, optionally from about 10,000 Da to 36,000 Da. In one embodiment, PEGin pegylated IL-2 is a substantially linear, straight-chain PEG.

In one embodiment, pegylated IL-2 of the invention will preferablyretain at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% of thebiological activity associated with the unmodified protein. In oneembodiment, biological activity refers to the ability to bind CD25.

The serum clearance rate of PEG-modified IL-2 may be decreased by about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90%, relative to theclearance rate of the unmodified IL-2. PEG-modified IL-2 may have acirculation half-life (t_(1/2)) which is enhanced relative to thehalf-life of unmodified IL-2. The half-life of PEG-IL-2, or variantsthereof, may be enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500%, oreven by 1000% relative to the half-life of unmodified IL-2. In someembodiments, the protein half-life is determined in vitro, such as in abuffered saline solution or in serum. In other embodiments, the proteinhalf-life is an in vivo circulation half-life, such as the half-life ofthe protein in the serum or other bodily fluid of an animal.

Other Extended-PK Groups

In some embodiments, the extended-PK group is a serum albumin, orfragments thereof. Methods of fusing serum albumin to proteins aredisclosed in, e.g., US2010/0144599, US2007/0048282, and US2011/0020345,which are herein incorporated by reference in their entirety. In someembodiments, the extended-PK group is human serum albumin. In someembodiments, the extended-PK group is transferrin.

In some embodiments, the extended-PK group is a serum albumin bindingprotein such as those described in US2005/0287153, US2007/0003549,US2007/0178082, US2007/0269422, US2010/0113339, WO2009/083804, andWO2009/133208, which are herein incorporated by reference in theirentirety.

In some embodiments, the extended-PK group is a serum immunoglobulinbinding protein such as those disclosed in US2007/0178082, which isherein incorporated by reference in its entirety.

In some embodiments, the extended-PK group is a fibronectin (Fn)-basedscaffold domain protein that binds to serum albumin, such as thosedisclosed in US2012/0094909, which is herein incorporated by referencein its entirety. Methods of making fibronectin-based scaffold domainproteins are also disclosed in US2012/0094909. A non-limiting example ofa Fn3-based extended-PK group is Fn3(HSA), i.e., a Fn3 protein thatbinds to human serum albumin.

Linkers

In some embodiments, the extended-PK group is optionally fused to IL-2via a linker. Linkers suitable for fusing the extended-PK group to IL-2are well known in the art, and are disclosed in, e.g., US2010/0210511US2010/0179094, and US2012/0094909, which are herein incorporated byreference in its entirety. Exemplary linkers include gly-ser polypeptidelinkers, glycine-proline polypeptide linkers, and proline-alaninepolypeptide linkers. In a preferred embodiment, the linker is a gly-serpolypeptide linker, i.e., a peptide that consists of glycine and serineresidues.

Exemplary gly-ser polypeptide linkers comprise the amino acid sequenceSer(Gly₄Ser)n. In one embodiment, n=1. In one embodiment, n=2. Inanother embodiment, n=3, i.e., Ser(Gly₄Ser)₃. In another embodiment,n=4, i.e., Ser(Gly₄Ser)4. In another embodiment, n=5. In yet anotherembodiment, n=6. In another embodiment, n=7. In yet another embodiment,n=8. In another embodiment, n=9. In yet another embodiment, n=10.Another exemplary gly-ser polypeptide linker comprises the amino acidsequence Ser(Gly₄Ser)n. In one embodiment, n=1. In one embodiment, n=2.In a preferred embodiment, n=3. In another embodiment, n=4. In anotherembodiment, n=5. In yet another embodiment, n=6. Another exemplarygly-ser polypeptide linker comprises (Gly₄Ser)n. In one embodiment, n=1.In one embodiment, n=2. In a preferred embodiment, n=3. In anotherembodiment, n=4. In another embodiment, n=5. In yet another embodiment,n=6. Another exemplary gly-ser polypeptide linker comprises (Gly₃Ser)n.In one embodiment, n=1. In one embodiment, n=2. In a preferredembodiment, n=3. In another embodiment, n=4. In another embodiment, n=5.In yet another embodiment, n=6.

Therapeutic Agents

The extended-PK IL-2 of the present invention can be used in conjunctionwith one or more therapeutic agents. In one embodiment, the therapeuticagent is a therapeutic antibody. In another embodiment, the therapeuticagent is a therapeutic protein. In another embodiment, the therapeuticagent is a small molecule. In another embodiment, the therapeutic agentis an antigen. In another embodiment, the therapeutic agent is apopulation of cells. In a preferred embodiment, the therapeutic agent isa therapeutic antibody.

Therapeutic Antibodies

In one embodiment, the methods of the invention can be performed withextended-PK IL-2 together with a therapeutic antibody.

Methods of producing antibodies, and antigen-binding fragments thereof,are well known in the art and are disclosed in, e.g., U.S. Pat. No.7,247,301, US2008/0138336, and U.S. Pat. No. 7,923,221, all of which areherein incorporated by reference in their entirety.

Therapeutic antibodies that can be used in the methods of the presentinvention include, but are not limited to, any of the art-recognizedanti-cancer antibodies that are approved for use, in clinical trials, orin development for clinical use. In some embodiments, more than oneanti-cancer antibody can be included in the combination therapy of thepresent invention.

Non-limiting examples of anti-cancer antibodies include the following,without limitation:

trastuzumab (HERCEPTIN™. by Genentech, South San Francisco, Calif.),which is used to treat HER-2/neu positive breast cancer or metastaticbreast cancer;

bevacizumab (AVASTIN™ by Genentech), which is used to treat colorectalcancer, metastatic colorectal cancer, breast cancer, metastatic breastcancer, non-small cell lung cancer, or renal cell carcinoma;

rituximab (RITUXAN™ by Genentech), which is used to treat non-Hodgkin'slymphoma or chronic lymphocytic leukemia;

pertuzumab (OMNITARG™ by Genentech), which is used to treat breastcancer, prostate cancer, non-small cell lung cancer, or ovarian cancer;

cetuximab (ERBITUX™ by ImClone Systems Incorporated, New York, N.Y.),which can be used to treat colorectal cancer, metastatic colorectalcancer, lung cancer, head and neck cancer, colon cancer, breast cancer,prostate cancer, gastric cancer, ovarian cancer, brain cancer,pancreatic cancer, esophageal cancer, renal cell cancer, prostatecancer, cervical cancer, or bladder cancer;

IMC-1C11 (ImClone Systems Incorporated), which is used to treatcolorectal cancer, head and neck cancer, as well as other potentialcancer targets;

tositumomab and tositumomab and iodine I¹³¹ (BEXXAR™ by CorixaCorporation, Seattle, Wash.), which is used to treat non-Hodgkin'slymphoma, which can be CD20 positive, follicular, non-Hodgkin'slymphoma, with and without transformation, whose disease is refractoryto Rituximab and has relapsed following chemotherapy;

In¹¹¹ ibirtumomab tiuxetan; Y⁹⁰ ibirtumomab tiuxetan; I¹¹¹ ibirtumomabtiuxetan and Y⁹⁰ ibirtumomab tiuxetan (ZEVALIN™ by Biogen Idec,Cambridge, Mass.), which is used to treat lymphoma or non-Hodgkin'slymphoma, which can include relapsed follicular lymphoma; relapsed orrefractory, low grade or follicular non-Hodgkin's lymphoma; ortransformed B-cell non-Hodgkin's lymphoma;

EMD 7200 (EMD Pharmaceuticals, Durham, N.C.), which is used for treatingfor treating non-small cell lung cancer or cervical cancer;

SGN-30 (a genetically engineered monoclonal antibody targeted to CD30antigen by Seattle Genetics, Bothell, Wash.), which is used for treatingHodgkin's lymphoma or non-Hodgkin's lymphoma;

SGN-15 (a genetically engineered monoclonal antibody targeted to aLewisγ-related antigen that is conjugated to doxorubicin by SeattleGenetics), which is used for treating non-small cell lung cancer;

SGN-33 (a humanized antibody targeted to CD33 antigen by SeattleGenetics), which is used for treating acute myeloid leukemia (AML) andmyelodysplastic syndromes (MDS);

SGN-40 (a humanized monoclonal antibody targeted to CD40 antigen bySeattle Genetics), which is used for treating multiple myeloma ornon-Hodgkin's lymphoma;

SGN-35 (a genetically engineered monoclonal antibody targeted to a CD30antigen that is conjugated to auristatin E by Seattle Genetics), whichis used for treating non-Hodgkin's lymphoma;

SGN-70 (a humanized antibody targeted to CD70 antigen by SeattleGenetics), that is used for treating renal cancer and nasopharyngealcarcinoma;

SGN-75 (a conjugate comprised of the SGN70 antibody and an Auristatinderivative by Seattle Genetics); and

SGN-17/19 (a fusion protein containing antibody and enzyme conjugated tomelphalan prodrug by Seattle Genetics), which is used for treatingmelanoma or metastatic melanoma.

It should be understood that the therapeutic antibodies to be used inthe methods of the present invention are not limited to those describedsupra. For example, the following approved therapeutic antibodies canalso be used in the methods of the invention: brentuximab vedotin(ADCETRIS™) for anaplastic large cell lymphoma and Hodgkin lymphoma,ipilimumab (MDX-101; YERVOY™) for melanoma, ofatumumab (ARZERRA™) forchromic lymphocytic leukemia, panitumumab (VECTIBIX™) for colorectalcancer, alemtuzumab (CAMPATH™) for chronic lymphocytic leukemia,ofatumumab (ARZERRA™) for chronic lymphocytic leukemia, gemtuzumabozogamicin (MYLOTARG™) for acute myelogenous leukemia.

Antibodies for use in the present invention can also target moleculesexpressed by immune cells, such as, but not limited to, tremelimumab(CP-675,206) and ipilimumab (MDX-010) which targets CTLA4 and has theeffect of tumor rejection, protection from rechallenge, and enhancedtumor-specific T cell responses; OX86 which targets OX40 and increasesantigen-specific CD8+ T cells at tumor sites and enhances tumorrejection; CT-011 which targets PD 1 and has the effect of maintainingand expanding tumor specific memory T cells and activates NK cells;BMS-663513 which targets CD137 and causes regression of establishedtumors, as well as the expansion and maintenance of CD8+ T cells, anddaclizumab (ZENAPAX™) which targets CD25 and causes transient depletionof CD4+CD25+FOXP3+Tregs and enhances tumor regression and increases thenumber of effector T cells. A more detailed discussion of theseantibodies can be found in, e.g., Weiner et al., Nature Rev. Immunol2010; 10:317-27.

The therapeutic antibody can be a fragment of an antibody; a complexcomprising an antibody; or a conjugate comprising an antibody. Theantibody can optionally be chimeric or humanized or fully human.

Therapeutic Proteins

In one embodiment, the methods of the invention include administrationof an extended-PK IL-2 and a therapeutic protein. Therapeutic proteinsthat are effective in treating cancer are well known in the art, and aredisclosed in, e.g., Dranoff et al., Nature Reviews Cancer 2004; 4:11-22.In one embodiment, the therapeutic protein is IL-7. In anotherembodiment, the therapeutic protein is GM-CSF.

The heterologous nucleic acid sequence generally encodes a diagnostic ortherapeutic polypeptide. In specific embodiments, the therapeuticpolypeptide or protein is a “suicide protein” that causes cell death byitself or in the presence of other compounds. A representative exampleof such a suicide protein is thymidine kinase of the herpes simplexvirus. Additional examples include thyrnidine kinase of varicella zostervirus, the bacterial gene cytosine deaminase (which converts5-fluorocytosine to the highly toxic compound 5-fluorouracil), p450oxidoreductase, carboxypeptidase G2, beta-glucuronidase,penicillin-V-amidase, penicillin-G-amidase, beta-lactamase,nitroreductase, carboxypeptidase A, linamarase (also referred to as.beta.-glucosidase), the E. coli gpt gene, and the E. coli Deo gene,although others are known in the art. In some embodiments, the suicideprotein converts a prodrug into a toxic compound. As used herein,“prodrug” means any compound useful in the methods of the presentinvention that can be converted to a toxic product, i.e. toxic to tumorcells. The prodrug is converted to a toxic product by the suicideprotein. Representative examples of such prodrugs include: ganciclovir,acyclovir, and FIAU(1-(2-deoxy-2-fluoro-.beta.-D-arabinofuranosyl)-5-iod-ouracil) forthymidine kinase; ifosfamide for oxidoreductase; 6-methoxypurinearabinoside for VZV-TK; 5-fluorocytosine for cytosine deaminase;doxorubicin for beta-glucuronidase; CB 1954 and nitrofurazone fornitroreductase; and N-(Cyanoacetyl)-L-phenylalanine orN-(3-chloropropionyl)-L-phenylalanine for carboxypeptidase A. Theprodrug may be administered readily by a person having ordinary skill inthis art. A person with ordinary skill would readily be able todetermine the most appropriate dose and route for the administration ofthe prodrug.

In some embodiments, a therapeutic protein or polypeptide, is a cancersuppressor, for example p53 or Rb, or a nude acid encoding such aprotein or polypeptide. Of course, those of skill know of a wide varietyof such cancer suppressors and how to obtain them and/or the nucleicacids encoding them.

Other examples of therapeutic proteins or polypeptides includepro-apoptotic therapeutic proteins and polypeptides, for example, p15,p16, or p21^(WAF-1).

Cytokines, and nucleic acid encoding them may also be used astherapeutic proteins and polypeptides. Examples include: GM-CSF(granulocyte macrophage colony stimulating factor); TNF-alpha (Tumornecrosis factor alpha); Interferons including, but not limited to,IFN-alpha and IFN-gamma; and Interleukins including, but not limited to,Interleukin-1 (IL-1), Interleukin-Beta (IL-beta), Interleukin-2 (IL-2),Interleukin-4 (IL-4), Interleukin-5 (IL-5), Interleukin-6 (IL-6),Interleukin-7 (IL-7), Interleukin-8 (IL-8), Interleukin-10 (IL-10),Interleukin-12 (IL-12), Interleukin-13 (IL-13), Interleukin-14 (IL-14),Interleukin-15 (IL-15), Interleukin-16 (IL-16), Interleukin-18 (IL-18),Interleukin-23 (IL-23), Interleukin-24 (IL-24), although otherembodiments are known in the art. In a preferred embodiment, thetherapeutic protein is IL-7.

Additional examples of cytocidal genes includes, but is not limited to,mutated cyclin G1 genes. By way of example, the cytocidal gene may be adominant negative mutation of the cyclin G1 protein (e.g., WO/01/64870).

It should be understood that the examples listed above are non-limiting,and that the methods of the present invention can be performed inconjunction with any art-recognized therapeutic protein that iseffective for treating cancer when used alone or as adjunctive therapy.

Small Molecules

In one embodiment, the methods of the invention include administrationof an extended-PK IL-2 and a small molecule. Small molecules that areeffective in treating cancer are well known in the art, and includeantagonists of factors that are involved in tumor growth, such as EGFR,ErbB2 (also known as Her2) ErbB3, ErbB4, or TNF. Non-limiting examplesinclude small molecule receptor tyrosine kinase inhibitors (RTKIs) thattarget one or more tyrosine kinase receptors, such as VEGF receptors,FGF receptors, EGF receptors and PDGF receptors. Many therapeutic smallmolecule RTKIs are known in the art, including, but are not limited to,vatalanib (PTK787), erlotinib (TARCEVA™), OSI-7904, ZD6474 (ZACTIMA™),ZD6126 (ANG453), ZD1839, sunitinib (SUTENT™), semaxanib (SU5416),AMG706, AG013736, Imatinib (GLEEVEC™), MLN-518, CEP-701, PKC-412,Lapatinib (GSK572016), VELCADE™, AZD2171, sorafenib (NEXAVAR™), XL880,and CHIR-265. Small molecule protein tyrosine phosphatase inhibitors,such as those disclosed in Jiang et al., Cancer Metastasis Rev. 2008;27:263-72 are also useful for practicing the methods of the invention.Such inhibitors can target, e.g., HSP2, PRL, PTP1B, or Cdc25phosphatases. Small molecules that target Bcl-2/Bcl-XL, such as thosedisclosed in US2008/0058322, are also useful for practicing the methodsof the present invention. Further exemplary small molecules for use inthe present invention are disclosed in Zhang et al. Nature Reviews:Cancer 2009; 9:28-39. In particular, chemotherapeutic agents that leadto immunogenic cell death such as anthracyclins (Kepp et al., Cancer andMetastasis Reviews 2011; 30:61-9) will be well suited for synergisticeffects with extended-PK IL-2.

It should be understood that the examples listed above are non-limiting,and that the methods of the present invention can be performed inconjunction with any art-recognized small molecule that is effective fortreating cancer when used alone or as adjunctive therapy.

Cancer Antigens

In another embodiment, the methods of the invention includeadministration of an extended-PK IL-2 and a cancer antigen, e.g., foruse as a cancer vaccine (see, e.g., Overwijk et al. Journal ofExperimental Medicine 2008; 198:569-80). Other cancer antigens that canbe used in vaccinations include, but are not limited to, (i)tumor-specific antigens, (ii) tumor-associated antigens, (iii) cellsthat express tumor-specific antigens, (iv) cells that expresstumor-associated antigens, (v) embryonic antigens on tumors, (vi)autologous tumor cells, (vii) tumor-specific membrane antigens, (viii)tumor-associated membrane antigens, (ix) growth factor receptors, (x)growth factor ligands, and (xi) any other type of antigen orantigen-presenting cell or material that is associated with a cancer.The cancer antigen may be an epithelial cancer antigen, (e.g., breast,gastrointestinal, lung), a prostate specific cancer antigen (PSA) orprostate specific membrane antigen (PSMA), a bladder cancer antigen, alung (e.g., small cell lung) cancer antigen, a colon cancer antigen, anovarian cancer antigen, a brain cancer antigen, a gastric cancerantigen, a renal cell carcinoma antigen, a pancreatic cancer antigen, aliver cancer antigen, an esophageal cancer antigen, a head and neckcancer antigen, or a colorectal cancer antigen. In another embodiment,the cancer antigen is a lymphoma antigen (e.g., non-Hodgkin's lymphomaor Hodgkin's lymphoma), a B-cell lymphoma cancer antigen, a leukemiaantigen, a myeloma (i.e., multiple myeloma or plasma cell myeloma)antigen, an acute lymphoblastic leukemia antigen, a chronic myeloidleukemia antigen, or an acute myelogenous leukemia antigen. It should beunderstood that the described cancer antigens are only exemplary andthat any cancer antigen can be targeted in the present invention.

In another embodiment, the cancer antigen is a mucin-1 protein orpeptide (MUC-1) that is found on all human adenocarcinomas: pancreas,colon, breast, ovarian, lung, prostate, head and neck, includingmultiple myelomas and some B cell lymphomas. Patients with inflammatorybowel disease, either Crohn's disease or ulcerative colitis, are at anincreased risk for developing colorectal carcinoma. MUC-1 is a type Itransmembrane glycoprotein. The major extracellular portion of MUC-1 hasa large number of tandem repeats consisting of 20 amino acids whichcomprise immunogenic epitopes. In some cancers it is exposed in anunglycosylated form that is recognized by the immune system (Gendler etal., J Biol Chem 1990; 265:15286-15293). In another embodiment, thecancer antigen is a mutated B-Raf antigen, which is associated withmelanoma and colon cancer. The vast majority of these mutationsrepresent a single nucleotide change of T-A at nucleotide 1796 resultingin a valine to glutamic acid change at residue 599 within the activationsegment of B-Raf. Raf proteins are also indirectly associated withcancer as effectors of activated Ras proteins, oncogenic forms of whichare present in approximately one-third of all human cancers. Normalnon-mutated B-Raf is involved in cell signaling, relaying signals fromthe cell membrane to the nucleus. The protein is usually only activewhen needed to relay signals. In contrast, mutant B-Raf has beenreported to be constantly active, disrupting the signaling relay (Mercerand Pritchard, Biochim Biophys Acta (2003) 1653(1):25-40; Sharkey etal., Cancer Res. (2004) 64(5):1595-1599).

In one embodiment, the cancer antigen is a human epidermal growth factorreceptor-2 (HER-2/neu) antigen. Cancers that have cells that overexpressHER-2/neu are referred to as HER-2/neu⁺ cancers. Exemplary HER-2/neu⁺cancers include prostate cancer, lung cancer, breast cancer, ovariancancer, pancreatic cancer, skin cancer, liver cancer (e.g.,hepatocellular adenocarcinoma), intestinal cancer, and bladder cancer.

HER-2/neu has an extracellular binding domain (ECD) of approximately 645aa, with 40% homology to epidermal growth factor receptor (EGFR), ahighly hydrophobic transmembrane anchor domain (TMD), and acarboxyterminal intracellular domain (ICD) of approximately 580 aa with80% homology to EGFR. The nucleotide sequence of HER-2/neu is availableat GENBANK™. Accession Nos. AH002823 (human HER-2 gene, promoter regionand exon 1); M16792 (human HER-2 gene, exon 4): M16791 (human HER-2gene, exon 3); M16790 (human HER-2 gene, exon 2); and M16789 (humanHER-2 gene, promoter region and exon 1). The amino acid sequence for theHER-2/neu protein is available at GENBANK™. Accession No. AAA58637.Based on these sequences, one skilled in the art could develop HER-2/neuantigens using known assays to find appropriate epitopes that generatean effective immune response. Exemplary HER-2/neu antigens includep369-377 (a HER-2/neu derived HLA-A2 peptide); dHER2 (CorixaCorporation); li-Key MHC class II epitope hybrid (Generex BiotechnologyCorporation); peptide P4 (amino acids 378-398); peptide P7 (amino acids610-623); mixture of peptides P6 (amino acids 544-560) and P7; mixtureof peptides P4, P6 and P7; HER2 [9₇₅₄]; and the like.

In one embodiment, the cancer antigen is an epidermal growth factorreceptor (EGFR) antigen. The EGFR antigen can be an EGFR variant 1antigen, an EGFR variant 2 antigen, an EGFR variant 3 antigen and/or anEGFR variant 4 antigen. Cancers with cells that overexpress EGFR arereferred to as EGFR cancers. Exemplary EGFR cancers include lung cancer,head and neck cancer, colon cancer, colorectal cancer, breast cancer,prostate cancer, gastric cancer, ovarian cancer, brain cancer andbladder cancer.

In one embodiment, the cancer antigen is a vascular endothelial growthfactor receptor (VEGFR) antigen. VEGFR is considered to be a regulatorof cancer-induced angiogenesis. Cancers with cells that overexpressVEGFR are called VEGFR⁺ cancers. Exemplary VEGFR⁺ cancers include breastcancer, lung cancer, small cell lung cancer, colon cancer, colorectalcancer, renal cancer, leukemia, and lymphocytic leukemia.

In one embodiment the cancer antigen is prostate-specific antigen (PSA)and/or prostate-specific membrane antigen (PSMA) that are prevalentlyexpressed in androgen-independent prostate cancers.

In another embodiment, the cancer antigen is Gp-100 Glycoprotein 100 (gp100) is a tumor-specific antigen associated with melanoma.

In one embodiment, the cancer antigen is a carcinoembryonic (CEA)antigen. Cancers with cells that overexpress CEA are referred to as CEA⁺cancers. Exemplary CEA⁺ cancers include colorectal cancer, gastriccancer and pancreatic cancer. Exemplary CEA antigens include CAP-1(i.e., CEA aa 571-579), CAP1-6D, CAP-2 (i.e., CEA aa 555-579), CAP-3(i.e., CEA aa 87-89), CAP-4 (CEA aa 1-11), CAP-5 (i.e., CEA aa 345-354),CAP-6 (i.e., CEA aa 19-28) and CAP-7.

In one embodiment, the cancer antigen is carbohydrate antigen 10.9 (CA19.9). CA 19.9 is an oligosaccharide related to the Lewis A blood groupsubstance and is associated with colorectal cancers.

In another embodiment, the cancer antigen is a melanoma cancer antigen.Melanoma cancer antigens are useful for treating melanoma. Exemplarymelanoma cancer antigens include MART-1 (e.g., MART-1 26-35 peptide,MART-1 27-35 peptide); MART-1/Melan A; pMel17; pMel17/gp100; gp100(e.g., gp 100 peptide 280-288, gp 100 peptide 154-162, gp 100 peptide457-467); TRP-1; TRP-2; NY-ESO-1; p16; beta-catenin; mum-1; and thelike.

In one embodiment, the cancer antigen is a mutant or wild type raspeptide. The mutant ras peptide can be a mutant K-ras peptide, a mutantN-ras peptide and/or a mutant H-ras peptide. Mutations in the rasprotein typically occur at positions 12 (e.g., arginine or valinesubstituted for glycine), 13 (e.g., asparagine for glycine), 61 (e.g.,glutamine to leucine) and/or 59. Mutant ras peptides can be useful aslung cancer antigens, gastrointestinal cancer antigens, hepatomaantigens, myeloid cancer antigens (e.g., acute leukemia,myelodysplasia), skin cancer antigens (e.g., melanoma, basal cell,squamous cell), bladder cancer antigens, colon cancer antigens,colorectal cancer antigens, and renal cell cancer antigens.

In another embodiment of the invention, the cancer antigen is a mutantand/or wildtype p53 peptide. The p53 peptide can be used as colon cancerantigens, lung cancer antigens, breast cancer antigens, hepatocellularcarcinoma cancer antigens, lymphoma cancer antigens, prostate cancerantigens, thyroid cancer antigens, bladder cancer antigens, pancreaticcancer antigens and ovarian cancer antigens.

The cancer antigen can be a cell, a protein, a peptide, a fusionprotein, DNA encoding a peptide or protein, RNA encoding a peptide orprotein, a glycoprotein, a lipoprotein, a phosphoprotein, acarbohydrate, a lipopolysaccharide, a lipid, a chemically linkedcombination of two or more thereof, a fusion or two or more thereof, ora mixture of two or more thereof. In another embodiment, the cancerantigen is a peptide comprising about 6 to about 24 amino acids; fromabout 8 to about 20 amino acids; from about 8 to about 12 amino acids;from about 8 to about 10 amino acids; or from about 12 to about 20 aminoacids. In one embodiment, the cancer antigen is a peptide having a MHCClass I binding motif or a MHC Class II binding motif. In anotherembodiment, the cancer antigen comprises a peptide that corresponds toone or more cytotoxic T lymphocyte (CTL) epitopes.

It should be understood that the examples listed above are non-limiting,and that the methods of the present invention can be performed inconjunction with any art-recognized cancer antigen that is known to beeffective, e.g., as a cancer vaccine, when used alone or as adjunctivetherapy.

Cell Therapy

In yet another embodiment, the methods of the invention includeadministration of an extended-PK IL-2 and a cell therapy. Cell therapiesthat are useful for treating cancer are well known and are disclosed in,e.g., U.S. Pat. No. 7,402,431. In a preferred embodiment, the celltherapy is T cell transplant. In a preferred method, T cells areexpanded ex vivo with IL-2 prior to transplantation into a subject.Methods for cell therapies are disclosed in, e.g., U.S. Pat. No.7,402,431, US2006/0057121, U.S. Pat. No. 5,126,132, U.S. Pat. No.6,255,073, U.S. Pat. No. 5,846,827, U.S. Pat. No. 6,251,385, U.S. Pat.No. 6,194,207, U.S. Pat. No. 5,443,983, U.S. Pat. No. 6,040,177, U.S.Pat. No. 5,766,920, and US2008/0279836.

It should be understood that the examples listed above are non-limiting,and that the methods of the present invention can be performed inconjunction with any art-recognized cell therapy that is effective fortreating cancer when used alone or as adjunctive therapy.

Methods of Making Extended-PK IL-2 Proteins

In some aspects, the extended-PK IL-2 proteins of the invention are madein transformed host cells using recombinant DNA techniques. To do so, arecombinant DNA molecule coding for the peptide is prepared. Methods ofpreparing such DNA molecules are well known in the art. For instance,sequences coding for the peptides could be excised from DNA usingsuitable restriction enzymes. Alternatively, the DNA molecule could besynthesized using chemical synthesis techniques, such as thephosphoramidate method. Also, a combination of these techniques could beused.

The invention also includes a vector capable of expressing the peptidesin an appropriate host. The vector comprises the DNA molecule that codesfor the peptides operatively linked to appropriate expression controlsequences. Methods of affecting this operative linking, either before orafter the DNA molecule is inserted into the vector, are well known.Expression control sequences include promoters, activators, enhancers,operators, ribosomal nuclease domains, start signals, stop signals, capsignals, polyadenylation signals, and other signals involved with thecontrol of transcription or translation.

The resulting vector having the DNA molecule thereon is used totransform an appropriate host. This transformation may be performedusing methods well known in the art.

Any of a large number of available and well-known host cells may be usedin the practice of this invention. The selection of a particular host isdependent upon a number of factors recognized by the art. These include,for example, compatibility with the chosen expression vector, toxicityof the peptides encoded by the DNA molecule, rate of transformation,ease of recovery of the peptides, expression characteristics, bio-safetyand costs. A balance of these factors must be struck with theunderstanding that not all hosts may be equally effective for theexpression of a particular DNA sequence. Within these generalguidelines, useful microbial hosts include bacteria (such as E. colisp.), yeast (such as Saccharomyces sp.) and other fungi, insects,plants, mammalian (including human) cells in culture, or other hostsknown in the art.

Next, the transformed host is cultured and purified. Host cells may becultured under conventional fermentation conditions so that the desiredcompounds are expressed. Such fermentation conditions are well known inthe art. Finally, the peptides are purified from culture by methods wellknown in the art.

The compounds may also be made by synthetic methods. For example, solidphase synthesis techniques may be used. Suitable techniques are wellknown in the art, and include those described in Merrifield (1973),Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.);Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985),Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid PhasePeptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), TheProteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins(3rd ed.) 2: 257-527. Solid phase synthesis is the preferred techniqueof making individual peptides since it is the most cost-effective methodof making small peptides. Compounds that contain derivatized peptides orwhich contain non-peptide groups may be synthesized by well-knownorganic chemistry techniques.

Other methods are of molecule expression/synthesis are generally knownin the art to one of ordinary skill.

Pharmaceutical Compositions and Modes of Administration

In certain embodiments, extended-PK IL-2 is administered together(simultaneously or sequentially) with one or more therapeutic agents,such as a therapeutic antibody. In certain embodiments, extended-PK IL-2is administered prior to the administration of one or more therapeuticagents, such as a therapeutic antibody. In certain embodiments,extended-PK IL-2 is administered concurrent with the administration ofone or more therapeutic agents, such as a therapeutic antibody. Incertain embodiments, extended-PK IL-2 is administered subsequent to theadministration of one or more therapeutic agents, such as a therapeuticantibody. In certain embodiments, the extended-PK IL-2 and one or moretherapeutic agents, such as a therapeutic antibody, are administeredsimultaneously. In other embodiments, the extended-PK IL-2 and one ormore therapeutic agents, such as a therapeutic antibody, areadministered sequentially. In yet other embodiments, the extended-PKIL-2 and one or more therapeutic agents, such as a therapeutic antibody,are administered within one, two, or three days of each other.

The one or more therapeutic agents may be those that serve as adjunctivetherapy for cancer, such as cytokines, chemotherapeutic agents, smallmolecules, antigens, or therapeutic antibodies, and are well known inthe art and discussed supra. Additional non-limiting examples ofadditional agents include GM-CSF (expands monocyte and neutrophilpopulation), IL-7 (important for generation and survival of memoryT-cells), interferon alpha, tumor necrosis factor alpha, IL-12, andtherapeutic antibodies, such as anti-PD-1, anti-PD-L, anti-CTLA4,anti-CD40, anti-OX45, and anti-CD137 antibodies. In some embodiments,the subject receives extended-PK IL-2 and one or more therapeutic agentsduring a same period of prevention, occurrence of a disorder, and/orperiod of treatment.

Pharmaceutical compositions of the invention can be administered incombination therapy, i.e., combined with other agents. Agents include,but are not limited to, in vitro synthetically prepared chemicalcompositions, antibodies, antigen binding regions, and combinations andconjugates thereof. In certain embodiments, an agent can act as anagonist, antagonist, allosteric modulator, or toxin.

In certain embodiments, the invention provides for separatepharmaceutical compositions comprising extended-PK IL-2 with apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative and/or adjuvant and another pharmaceutical compositioncomprising one or more therapeutic agents, such as a therapeuticantibody, with a pharmaceutically acceptable diluent, carrier,solubilizer, emulsifier, preservative and/or adjuvant.

In certain embodiments, the invention provides for pharmaceuticalcompositions comprising extended-PK IL-2 and one or more therapeuticagents, such as a therapeutic antibody, and a therapeutically effectiveamount of at least one additional therapeutic agent, together with apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative and/or adjuvant, and another pharmaceutical compositioncomprises one or more therapeutic agents, e.g., a therapeutic antibody,together with a pharmaceutically acceptable diluent, carrier,solubilizer, emulsifier, preservative and/or adjuvant. In someembodiments, each of the agents, e.g., extended-PK IL-2, therapeuticantibody, and the additional therapeutic agent can be formulated asseparate compositions.

In certain embodiments, acceptable formulation materials preferably arenontoxic to recipients at the dosages and concentrations employed. Insome embodiments, the formulation material(s) are for s.c. and/or I.V.administration. In certain embodiments, the pharmaceutical compositioncan contain formulation materials for modifying, maintaining orpreserving, for example, the pH, osmolarity, viscosity, clarity, color,isotonicity, odor, sterility, stability, rate of dissolution or release,adsorption or penetration of the composition. In certain embodiments,suitable formulation materials include, but are not limited to, aminoacids (such as glycine, glutamine, asparagine, arginine or lysine);antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite orsodium hydrogen-sulfite); buffers (such as borate, bicarbonate,Tris-HCl, citrates, phosphates or other organic acids); bulking agents(such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants.(Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed.,Mack Publishing Company (1995). In some embodiments, the formulationcomprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or 10 mM NAOAC, pH5.2, 9% Sucrose.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, Remington's Pharmaceutical Sciences, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance ofextended-PK IL-2 and one or more therapeutic agents.

In certain embodiments, the primary vehicle or carrier in apharmaceutical composition can be either aqueous or non-aqueous innature. For example, in certain embodiments, a suitable vehicle orcarrier can be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. In someembodiments, the saline comprises isotonic phosphate-buffered saline. Incertain embodiments, neutral buffered saline or saline mixed with serumalbumin are further exemplary vehicles. In certain embodiments,pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, oracetate buffer of about pH 4.0-5.5, which can further include sorbitolor a suitable substitute therefore. In certain embodiments, acomposition comprising extended-PK IL-2 and one or more therapeuticantibodies, with or without one or more therapeutic agents, can beprepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (Remington'sPharmaceutical Sciences, supra) in the form of a lyophilized cake or anaqueous solution. Further, in certain embodiments, a compositioncomprising extended-PK IL-2 and one or more therapeutic antibodies, withor without one or more therapeutic agents, can be formulated as alyophilizate using appropriate excipients such as sucrose.

In certain embodiments, the pharmaceutical composition can be selectedfor parenteral delivery. In certain embodiments, the compositions can beselected for inhalation or for delivery through the digestive tract,such as orally. The preparation of such pharmaceutically acceptablecompositions is within the ability of one skilled in the art.

In certain embodiments, the formulation components are present inconcentrations that are acceptable to the site of administration. Incertain embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

In certain embodiments, when parenteral administration is contemplated,a therapeutic composition can be in the form of a pyrogen-free,parenterally acceptable aqueous solution comprising a desiredextended-PK IL-2 and one or more therapeutic agents, such as atherapeutic antibody, in a pharmaceutically acceptable vehicle. Incertain embodiments, a vehicle for parenteral injection is steriledistilled water in which extended-PK IL-2 and one or more therapeuticagents, such as a therapeutic antibody, are formulated as a sterile,isotonic solution, properly preserved. In certain embodiments, thepreparation can involve the formulation of the desired molecule with anagent, such as injectable microspheres, bio-erodible particles,polymeric compounds (such as polylactic acid or polyglycolic acid),beads or liposomes, that can provide for the controlled or sustainedrelease of the product which can then be delivered via a depotinjection. In certain embodiments, hyaluronic acid can also be used, andcan have the effect of promoting sustained duration in the circulation.In certain embodiments, implantable drug delivery devices can be used tointroduce the desired molecule.

In certain embodiments, a pharmaceutical composition can be formulatedfor inhalation. In certain embodiments, extended-PK IL-2 and one or moretherapeutic agents, such as a therapeutic antibody, can be formulated asa dry powder for inhalation. In certain embodiments, an inhalationsolution comprising extended-PK IL-2 and one or more therapeutic agents,such as a therapeutic antibody, can be formulated with a propellant foraerosol delivery. In certain embodiments, solutions can be nebulized.Pulmonary administration is further described in PCT application no.PCT/US94/001875, which describes pulmonary delivery of chemicallymodified proteins.

In certain embodiments, it is contemplated that formulations can beadministered orally. In certain embodiments, extended-PK IL-2 and one ormore therapeutic agents, such as a therapeutic antibody, that isadministered in this fashion can be formulated with or without thosecarriers customarily used in the compounding of solid dosage forms suchas tablets and capsules. In certain embodiments, a capsule can bedesigned to release the active portion of the formulation at the pointin the gastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. In certain embodiments, at leastone additional agent can be included to facilitate absorption ofextended-PK IL-2 and one or more therapeutic agents, such as atherapeutic antibody. In certain embodiments, diluents, flavorings, lowmelting point waxes, vegetable oils, lubricants, suspending agents,tablet disintegrating agents, and binders can also be employed.

In certain embodiments, a pharmaceutical composition can involve aneffective quantity of extended-PK IL-2 and one or more therapeuticagents, such as a therapeutic antibody, in a mixture with non-toxicexcipients which are suitable for the manufacture of tablets. In certainembodiments, by dissolving the tablets in sterile water, or anotherappropriate vehicle, solutions can be prepared in unit-dose form. Incertain embodiments, suitable excipients include, but are not limitedto, inert diluents, such as calcium carbonate, sodium carbonate orbicarbonate, lactose, or calcium phosphate; or binding agents, such asstarch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving extended-PK IL-2 and one ormore therapeutic agents, such as a therapeutic antibody, in sustained-or controlled-delivery formulations. In certain embodiments, techniquesfor formulating a variety of other sustained- or controlled-deliverymeans, such as liposome carriers, bio-erodible microparticles or porousbeads and depot injections, are also known to those skilled in the art.See for example, PCT Application No. PCT/US93/00829 which describes thecontrolled release of porous polymeric microparticles for the deliveryof pharmaceutical compositions. In certain embodiments,sustained-release preparations can include semipermeable polymermatrices in the form of shaped articles, e.g. films, or microcapsules.Sustained release matrices can include polyesters, hydrogels,polylactides (U.S. Pat. No. 3,773,919 and EP 058,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22:547-556 (1983)), poly (2-hydroxyethyl-methacrylate) (Langer et al.,J. Biomed. Mater. Res., 15:167-277 (1981) and Langer, Chem. Tech.,12:98-105 (1982)), ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (EP 133,988). In certain embodiments,sustained release compositions can also include liposomes, which can beprepared by any of several methods known in the art. See, e.g., Eppsteinet al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP088,046 and EP 143,949.

The pharmaceutical composition to be used for in vivo administrationtypically is sterile. In certain embodiments, this can be accomplishedby filtration through sterile filtration membranes. In certainembodiments, where the composition is lyophilized, sterilization usingthis method can be conducted either prior to or following lyophilizationand reconstitution. In certain embodiments, the composition forparenteral administration can be stored in lyophilized form or in asolution. In certain embodiments, parenteral compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

In certain embodiments, once the pharmaceutical composition has beenformulated, it can be stored in sterile vials as a solution, suspension,gel, emulsion, solid, or as a dehydrated or lyophilized powder. Incertain embodiments, such formulations can be stored either in aready-to-use form or in a form (e.g., lyophilized) that is reconstitutedprior to administration.

In certain embodiments, kits are provided for producing a single-doseadministration unit. In certain embodiments, the kit can contain both afirst container having a dried protein and a second container having anaqueous formulation. In certain embodiments, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are included.

In certain embodiments, the effective amount of a pharmaceuticalcomposition comprising extended-PK IL-2 and one or more pharmaceuticalcompositions comprising therapeutic agents, such as a therapeuticantibody, to be employed therapeutically will depend, for example, uponthe therapeutic context and objectives. One skilled in the art willappreciate that the appropriate dosage levels for treatment, accordingto certain embodiments, will thus vary depending, in part, upon themolecule delivered, the indication for which extended-PK IL-2 and one ormore therapeutic agents such as a therapeutic antibody, are being used,the route of administration, and the size (body weight, body surface ororgan size) and/or condition (the age and general health) of thepatient. In certain embodiments, the clinician can titer the dosage andmodify the route of administration to obtain the optimal therapeuticeffect. In certain embodiments, a typical dosage can range from about0.1 μg/kg to up to about 100 mg/kg or more, depending on the factorsmentioned above. In certain embodiments, the dosage can range from 0.1μg/kg up to about 100 mg/kg; or 1 μg/kg up to about 100 mg/kg; or 5μg/kg up to about 100 mg/kg.

In certain embodiments, the frequency of dosing will take into accountthe pharmacokinetic parameters of extended-PK IL-2 and one or moretherapeutic agents, such as a therapeutic antibody, in the formulationused. In certain embodiments, a clinician will administer thecomposition until a dosage is reached that achieves the desired effect.In certain embodiments, the composition can therefore be administered asa single dose, or as two or more doses (which may or may not contain thesame amount of the desired molecule) over time, or as a continuousinfusion via an implantation device or catheter. Further refinement ofthe appropriate dosage is routinely made by those of ordinary skill inthe art and is within the ambit of tasks routinely performed by them. Incertain embodiments, appropriate dosages can be ascertained through useof appropriate dose-response data.

In certain embodiments, the route of administration of thepharmaceutical composition is in accord with known methods, e.g. orally,through injection by intravenous, intraperitoneal, intracerebral(intra-parenchymal), intracerebroventricular, intramuscular,subcutaneously, intra-ocular, intraarterial, intraportal, orintralesional routes; by sustained release systems or by implantationdevices. In certain embodiments, the compositions can be administered bybolus injection or continuously by infusion, or by implantation device.In certain embodiments, individual elements of the combination therapymay be administered by different routes.

In certain embodiments, the composition can be administered locally viaimplantation of a membrane, sponge or another appropriate material ontowhich the desired molecule has been absorbed or encapsulated. In certainembodiments, where an implantation device is used, the device can beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule can be via diffusion, timed-release bolus, or continuousadministration.

In certain embodiments, it can be desirable to use a pharmaceuticalcomposition comprising extended-PK IL-2 and one or more therapeuticagents, such as a therapeutic antibody in an ex vivo manner. In suchinstances, cells, tissues and/or organs that have been removed from thepatient are exposed to a pharmaceutical composition comprisingextended-PK IL-2 and one or more therapeutic agents, such as atherapeutic antibody, after which the cells, tissues and/or organs aresubsequently implanted back into the patient.

In certain embodiments, extended-PK IL-2 and one or more therapeuticagents, such as a therapeutic antibody, can be delivered by implantingcertain cells that have been genetically engineered, using methods suchas those described herein, to express and secrete the polypeptides. Incertain embodiments, such cells can be animal or human cells, and can beautologous, heterologous, or xenogeneic. In certain embodiments, thecells can be immortalized. In certain embodiments, in order to decreasethe chance of an immunological response, the cells can be encapsulatedto avoid infiltration of surrounding tissues. In certain embodiments,the encapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow the release of the proteinproduct(s) but prevent the destruction of the cells by the patient'simmune system or by other detrimental factors from the surroundingtissues.

Kits

A kit can include extended-PK IL-2 and one or more therapeutic agents,such as a therapeutic antibody, disclosed herein and instructions foruse. The kits may comprise, in a suitable container, extended-PK IL-2and one or more therapeutic agents, such as a therapeutic antibody, oneor more controls, and various buffers, reagents, enzymes and otherstandard ingredients well known in the art.

The container can include at least one vial, well, test tube, flask,bottle, syringe, or other container means, into extended-PK IL-2 and oneor more therapeutic agents, such as a therapeutic antibody, may beplaced, and in some instances, suitably aliquoted. Where an additionalcomponent is provided, the kit can contain additional containers intowhich this component may be placed. The kits can also include a meansfor containing extended-PK IL-2 and one or more therapeutic agents, suchas a therapeutic antibody, and any other reagent containers in closeconfinement for commercial sale. Such containers may include injectionor blow-molded plastic containers into which the desired vials areretained. Containers and/or kits can include labeling with instructionsfor use and/or warnings.

Methods of Treatment

The extended-PK IL-2 and one or more therapeutic agents, such as atherapeutic antibody, and/or nucleic acids expressing them, are usefulfor treating a disorder associated with abnormal apoptosis or adifferentiative process (e.g., cellular proliferative disorders orcellular differentiative disorders, such as cancer). Non-limitingexamples of cancers that are amenable to treatment with the methods ofthe present invention are described below. Extended-PK IL-2, wherein theIL-2 moiety is wild-type IL-2, is the preferred molecule for use in themethods of the invention.

Examples of cellular proliferative and/or differentiative disordersinclude cancer (e.g., carcinoma, sarcoma, metastatic disorders orhematopoietic neoplastic disorders, e.g., leukemias). A metastatic tumorcan arise from a multitude of primary tumor types, including but notlimited to those of prostate, colon, lung, breast and liver.Accordingly, the compositions of the present invention (e.g.,extended-PK IL-2 and one or more therapeutic agents, such as atherapeutic antibody and/or the nucleic acid molecules that encode them)can be administered to a patient who has cancer. Extended-PK IL-2 andone or more therapeutic agents, such as a therapeutic antibody, can beused to treat a patient (e.g., a patient who has cancer) prior to, orsimultaneously with, the administration of ex vivo expanded T cells.

As used herein, we may use the terms “cancer” (or “cancerous”),“hyperproliferative,” and “neoplastic” to refer to cells having thecapacity for autonomous growth (i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth). Hyperproliferativeand neoplastic disease states may be categorized as pathologic (i.e.,characterizing or constituting a disease state), or they may becategorized as non-pathologic (i.e., as a deviation from normal but notassociated with a disease state). The terms are meant to include alltypes of cancerous growths or oncogenic processes, metastatic tissues ormalignantly transformed cells, tissues, or organs, irrespective ofhistopathologic type or stage of invasiveness. “Pathologichyperproliferative” cells occur in disease states characterized bymalignant tumor growth. Examples of non-pathologic hyperproliferativecells include proliferation of cells associated with wound repair.

The term “cancer” or “neoplasm” are used to refer to malignancies of thevarious organ systems, including those affecting the lung, breast,thyroid, lymph glands and lymphoid tissue, gastrointestinal organs, andthe genitourinary tract, as well as to adenocarcinomas which aregenerally considered to include malignancies such as most colon cancers,renal-cell carcinoma, prostate cancer and/or testicular tumors,non-small cell carcinoma of the lung, cancer of the small intestine andcancer of the esophagus.

The term “carcinoma” is art recognized and refers to malignancies ofepithelial or endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. The mutant IL-2 polypeptidescan be used to treat patients who have, who are suspected of having, orwho may be at high risk for developing any type of cancer, includingrenal carcinoma or melanoma, or any viral disease. Exemplary carcinomasinclude those forming from tissue of the cervix, lung, prostate, breast,head and neck, colon and ovary. The term also includes carcinosarcomas,which include malignant tumors composed of carcinomatous and sarcomatoustissues. An “adenocarcinoma” refers to a carcinoma derived fromglandular tissue or in which the tumor cells form recognizable glandularstructures.

Additional examples of proliferative disorders include hematopoieticneoplastic disorders. As used herein, the term “hematopoietic neoplasticdisorders” includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. Preferably, the diseases arisefrom poorly differentiated acute leukemias (e.g., erythroblasticleukemia and acute megakaryoblastic leukemia). Additional exemplarymyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit. Rev. inOncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

It will be appreciated by those skilled in the art that amounts for eachof the extended-PK IL-2 and the one or more therapeutic agents, such asa therapeutic antibody, that are sufficient to reduce tumor growth andsize, or a therapeutically effective amount, will vary not only on theparticular compounds or compositions selected, but also with the routeof administration, the nature of the condition being treated, and theage and condition of the patient, and will ultimately be at thediscretion of the patient's physician or pharmacist. The length of timeduring which the compounds used in the instant method will be givenvaries on an individual basis.

It will be appreciated by those skilled in the art that reference hereinto treatment extends to prophylaxis as well as the treatment of thenoted cancers and symptoms.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for. The practice of the presentinvention will employ, unless otherwise indicated, conventional methodsof protein chemistry, biochemistry, recombinant DNA techniques andpharmacology, within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., T. E. Creighton, Proteins:Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition);Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition,1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., AcademicPress, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton,Pa.: Mack Publishing Company, 1990); Carey and Sundberg Advanced OrganicChemistry 3^(rd) Ed. (Plenum Press) Vols A and B (1992). Moreover, whilethe examples below employ extended-PK IL-2 of mouse origin, it should beunderstood that corresponding human extended-PK IL-2 can be readilygenerated by those of ordinary skill in the art using methods describedsupra, and used in the methods of the present invention.

Example 1 Materials and Methods Model of Mouse IL-2/IL-2R Complex

The homology model of the mouse IL-2/IL-2 receptor complex was builtusing SWISS-MODEL (Schwede et al., Nucleic Acids Research 2003;31:3381-5), by threading the mouse protein sequences into the crystalstructure the human IL-2/IL-2 receptor complex (Wang et al., Science2005; 310:1159-63).

IL-2 Affinity Maturation

A plasmid containing the coding sequence of murine IL-2, pORF-mIL2, waspurchased from InvivoGen. The IL-2 coding sequence was amplified by PCRusing primers with flanking NheI and BamHI restriction sites and clonedinto pCTCON2 by ligation. The resulting plasmid, pCT-mIL2, wastransformed into EBY100 using the Frozen-EZ Yeast Transformation II Kit(Zymo Research) according to the manufacturer's instructions. IL-2display was verified using biotinylated rat anti-HA, clone 3F10, (Roche)and chicken anti-c-myc (Invitrogen). Proper IL-2 folding was confirmedby labeling with His-tagged recombinant mouse IL-2Rα/CD25 (R&D Systems)and rabbit polyclonal anti-6×His tag antibody (Abcam) fluorescentlylabeled using the Alexa Fluor 647 Monoclonal Antibody Labeling Kit(Invitrogen).

In general, affinity maturation of IL-2 was performed as previouslydescribed (Chao et al. Nat Protocols 2006 1:755-768; Rao et al., MolPharmacol 2004; 66:864-9; U.S. Pat. No. 7,569,215; U.S. Pat. No.7,953,610). Briefly, the IL-2 coding sequence was mutagenized byerror-prone PCR under conditions predicted to yield predominantlymutants with one or two amino acid changes per protein. Library insertsand digested pCTCON2 backbone were transformed into competent EBY100 byelectroporation. Maximal library diversity was determined by platingserial dilutions of the library immediately after YPD outgrowth ontoSD-CAA plates. Plasmids were retrieved from libraries using the ZymoprepYeast Plasmid Miniprep I Kit (Zymo Research); single clones wereobtained after transformation into E. coli strain XL1-Blue (Strategene).

FACS screening of libraries 1.0 to 5.2 were conducted atnear-equilibrium labeling with soluble CD25 (decreasing from 25 to 1 nM)in PBS with 0.1 wt % BSA. FACS screening of libraries 6.0 and 6.1 wereconducted with kinetic competition, using library 5.2 mutants generatedby reducing the disulfide bond between Aga1p and Aga2p on the surface ofdisplaying yeast with TCEP.

Selective-reversion mutants of library 6.2 clones were created bysite-directed mutagenesis using oligonucleotides (Integrated DNATechnologies) containing the desired mutations.

The equilibrium dissociation constant of any particular clone wasdetermined essentially as described (75). The number of cells and volumeper sample were selected to ensure at least 10-fold excess of CD25relative to yeast-displayed IL-2 or mutant. For picomolar affinitymutants of library 6.2, non-displaying EBY100 cells were spiked in at90:10 to facilitate pelleting the low numbers of displaying cells. Theequilibrium dissociation constant K_(d) was determined by fitting CD25concentration and flow cytometry fluorescence data to the monovalentbinding isotherm:

MFU _(tot) =MFU _(min)+(MFU _(range) ×[CD25])/([CD25]+K _(d))

where MFU_(tot)=total mean fluorescence,

-   -   [CD25]=concentration of CD25, and        MFU_(min), MFU_(range), and K_(d) are constants

Flow Cytometry

DNA encoding IL-2 and QQ 6.2-10 were each subcloned into a pRS316-basedsecretion plasmid containing the galactose-inducible GAL 1-10 promoter,the engineered secretion leader αpp8 (77), and a C-terminal FLAG tag.Each secretion plasmid was co-transformed with pRS314 into yeast strainYVH10 (78). After growth to mid-log phase (OD₆₀₀˜5) at 30° C. inSD-SCAA, protein expression was induced by medium change to SG-SCAA,with BSA as carrier. Cell culture supernatants were harvested 3 dayspost induction and concentrated by ultrafiltration using Ultracel YM-10membranes (Millipore). IL-2 and QQ 6.2-10 were purified byimmunoaffinity chromatography using anti-FLAG M2 Affinity Gel (Sigma)and size exclusion chromatography using a Superdex 200 column (AmershamBiosciences). Protein purity was verified by silver staining of SDS-PAGEgels using SilverXpress Silver Staining Kit (Invitrogen) and westernblotting using anti-FLAG M2 peroxidase conjugate (Sigma-Aldrich).Protein concentrations were determined by quantitative western blotting,using Amino-terminal FLAG-BAP Fusion Protein (Sigma-Aldrich) asstandard.

Protein Production and Purification

DNA encoding IL-2 and QQ 6.2-10 were each subcloned into a pRS316-basedsecretion plasmid containing the galactose-inducible GAL 1-10 promoter,the engineered secretion leader αpp8 (77), and a C-terminal FLAG tag.Each secretion plasmid was co-transformed with pRS314 into yeast strainYVH10 (78). After growth to mid-log phase (OD₆₀₀˜5) at 30° C. inSD-SCAA, protein expression was induced by medium change to SG-SCAA,with BSA as carrier. Cell culture supernatants were harvested 3 dayspost induction and concentrated by ultrafiltration using Ultracel YM-10membranes (Millipore). IL-2 and QQ 6.2-10 were purified byimmunoaffinity chromatography using anti-FLAG M2 Affinity Gel (Sigma)and size exclusion chromatography using a Superdex 200 column (AmershamBiosciences). Protein purity was verified by silver staining of SDS-PAGEgels using SilverXpress Silver Staining Kit (Invitrogen) and westernblotting using anti-FLAG M2 peroxidase conjugate (Sigma-Aldrich).Protein concentrations were determined by quantitative western blotting,using Amino-terminal FLAG-BAP Fusion Protein (Sigma-Aldrich) asstandard.

Cell Culture

CTLL-2 cells were cultured in RPMI-1640 supplemented with FBS,L-glutamine, penicillin streptomycin (Invitrogen). For maintenance,cells were passaged every other day to 100,000 cells/ml in mediasupplemented with 100 pM wild-type mouse IL-2 (R&D Systems). For surfacepersistence and viability assays, cells were passaged to 200,000cells/ml and cultured in cytokine-free medium for 12 hours prior topulse with yeast-secreted IL-2 or QQ 6.2-10. Thirty minutes after IL-2or QQ 6.2-10 addition, cells were pelleted and resuspended incytokine-free medium. Cell-surface cytokine levels were determined bylabeling with M2 mouse anti-FLAG antibody (Sigma-Aldrich). Cell cultureviability was determined using the CellTiter-Glo Luminescent CellViability Assay (Promega), according to manufacturer's instructions.

Design and Characterization of Non-CD25 Binding IL-2 Mutants

Mutations at amino acid positions 76, 82, and 121 of IL-2 wereintroduced into pCT-mIL2 by PCR using oligonucleotides (Integrated DNATechnologies) containing the desired mutations. EBY100 yeast harboringeach of these clones were grown and induced as described (Chao et al.Nat Protocols 2006; 1(2):755-768). CD25 binding capacity was determinedby labeling induced yeast with 50 nM soluble murine CD25 (R&D Systems).Proper protein folding was determined by labeling yeast displaying IL-2variants before and after thermal denaturation with anti-mouse IL-2antibodies. Yeast displayed IL-2 variants were thermally denatured byincubating induced yeast at 85° C. for 30 minutes. Anti-mouse IL-2antibodies used were S4B6 (gift of Jianzhu Chen Lab, MIT), JES6-1A12,and JES6-5H4 (eBioscience).

Antibody binding both before and after thermal denaturation of IL-2mutant indicates antibody is non-conformation specific (JES6-1A12). Lossof antibody binding after thermal denaturation indicates antibody isconformation-specific (S4B6 and JES6-5H4). IL-2 variants E76A and E76Gwere detected by S4B6 before but not after thermal denaturation,suggesting they are properly folded proteins that lack CD25 bindingcapacity. Proper folding of E76A and E76G is further validated by theirstimulation of CTLL-2 growth in vitro as well as E76G's stimulation CD8+T cell and NK cell expansion in vivo.

Pharmacokinetics

Fc/IL-2 fusions were labeled with IRDye 800CW (LI-COR Biosciences)according to manufacturer's instructions. Unreacted dye was removedusing Zeba desalting columns (Thermo Scientific). Labeled proteins, 50μg in 100 μl PBS per dose, were administered intravenously byretro-orbital injection. At time=0, 0.5, 1, 3, 5, 8, 24, 48, and 96hours after injection, blood samples were collected from the tip of thetail into heparin-coated capillary tubes (VWR International). Sampleswere stored at 4° C., protected from light, until analysis.

On day of analysis, blood samples were centrifuged (15 min at 12000 rpmat 4° C.) to remove cellular components. The plasma was transferred tofresh capillary tubes and scanned using an Odyssey Infrared ImagingSystem (L1-COR Biosciences). Signal was acquired in the 800 nm channel,which corresponds to serum levels of Fc/IL-2 fusions. Using the imageprocessing program ImageJ (US National Institutes of Health), eachsample was approximated as a line of width 2 and the mean intensityalong was line was determined. The mean fluorescence intensities werethen fit to a biexponential:

MFI(t)=Ae ^(−αt) +Be ^(−βt)

where MFI=mean fluorescence intensity,

-   -   t=time, and        -   A, α, B, and β are constants

Fc/IL-2 fusion proteins were administered intravenously by retro-orbitalinjection. Four days post injection, animals were euthanized by CO2asphyxiation. Animals were weighted on a scale.

Pulmonary Wet Weight

Lungs from mice injected with Fc/IL-2 fusions were extracted and placedinto scintillation vials. Samples were weighed, frozen in liquidnitrogen, and lyophilized for 48 hours at room temperature under vacuum.Pulmonary wet weight was calculated by subtracting the sample weightafter lyophilization from the initial sample weight.

Flow Cytometry

Single-cell suspensions of spleen were prepared by rubbing spleensbetween two frosted microscope slides. Red blood cells were lysed withammonium chloride and passed through mesh filters to remove hair anddebris. Antibodies against CD3, CD4, CD8, CD25, NK1.1, Foxp3, CellTraceCalcein Violet (Invitrogen) were used.

Samples were analyzed using a LSR II flow cytometer using FACS Divasoftware (BD Biosciences). Flow cytometry data was analyzed using FlowJosoftware (Tree Star, Inc.). Total cell number per spleen was calculatedbased on number of cells processed by cytometer and fraction ofsplenocyte suspension analyzed.

Histology

Kidneys, livers, lungs, and spleens were fixed in 10% formalinovernight, embedded in paraffin, sectioned at 6 μm, and stained withhematoxylin and eosin. Tumors were cut in half: one half was fixed andstained as organs above; the other was embedded in O.C.T. media, andfrozen in isopentane in liquid nitrogen, and sectioned at 6 μm.

Example 2 Generation of High Affinity CD25-Binding IL-2 Mutants

Mouse IL-2 was affinity matured with error-prone PCR and yeast surfacedisplay to obtain high affinity CD25-binding IL-2 mutants. Themutagenesis approach and affinity maturation progress was determined byreferencing a model of the mouse IL-2/IL-2R complex based on the crystalstructure of the human IL-2/IL-2R complex. Error-prone PCR conditions(nucleotide analogue concentration and amplification cycle number) werechosen such as to produce one to two amino acid mutations per gene,distributed throughout the entire IL-2 gene.

A yeast surface display library was labeled with soluble CD25 andscreened six times for higher affinity clones by FACS. Sequences from aselection of clones indicated accumulation of mutants that encodeproline or threonine at position 126, which is serine in wild-type mouseIL-2. Notably, position 126 is proline or threonine in many other animalspecies. According to the model of the IL-2/IL-2 receptor complex, thisposition locates to the interface with CD25. Further affinity maturationof S126P and S126T IL-2, which bound to CD25 with an affinity 2 to3-fold higher than wild-type IL-2, led to the generation of IL-2 mutantswith 500-fold affinity improvement over wild-type IL-2. When thesemutants were sequenced, their mutations were found to locate to twodifference faces of IL-2, that in potential contact with CD25 and thatin potential contact with IL-2Rβ.

To avoid disrupting the interaction with IL-2Rβ, putative IL-2Rβ-bindingmutations were mutated so as to revert the mutations back to thewild-type amino acid residues by site-directed mutagenesis. The mutantsand their sequences are shown in FIG. 1. These reversion mutantsretained high CD25 binding affinity (FIG. 2). For convenience,high-affinity CD25-binding QQ 6.2-10 (“QQ6210”) was used in furtherexperiments.

Example 3 Generation of a Non-CD25-Binding IL-2 Mutant

Inspection of the mouse IL-2/IL-2 receptor complex revealed three aminoacid residues in intimate contact with CD25: E76, H82, and Q121 (FIG.3). To disrupt CD25 binding, each of these residues was mutated to oneof four alternative amino acids that differ from the wild-type in size,hydrophobicity, or charge. These 12 mutants were displayed on thesurface of yeast and tested for CD25 binding by labeling with 5 or 50 nMsoluble CD25.

TABLE 1 Mutations E76 --> R, F, A, G H82 --> E, S, A, G Q121 --> R, S,A, G

While all H82 and Q121 mutants retained CD25 binding, no CD25 bindingwas detected for E76 mutants (FIG. 4). Labeling of E76 mutants withconformation-specific anti-mouse IL-2 antibodies, with or withoutthermal denaturation, suggested that E76A and E76G are well-foldedproteins with no detectable binding at 50 nM soluble CD25 (FIG. 4).

Example 4 Fc/IL-2 and Mutants

A vector encoding the heavy chain of a mouse IgG2a from C57BL/6 mice wasprovided by J. Ravetch (The Rockefeller University). A fragment encodingthe hinge, C_(H)2, and C_(H)3 domains was cloned into the gWIZ vector(Genlantis) from PstI to SalI sites. Mouse IL-2 with a 6×His tag wassubsequently cloned into the vector C-terminal to Fc. To enableexpression of monovalent Fc/IL-2, a vector encoding the Fc with a FLAGtag was also constructed. Notably, a D265A mutation was introduced intothe Fc coding sequence to reduce effector function (i.e., to reduce ADCCand CDC) as disclosed in Baudino et al. (J Immunol 2008; 181:6664-9).DNA sequences were confirmed by DNA sequencing. Plasmid DNA wastransformed into XL1-Blue for amplification. DNA was purified from cellsusing PureLink HiPure Maxiprep Kit (Invitrogen) and sterile filtered.

HEK293 cells (Invitrogen) were cultured according to manufacturer'sinstructions. gWIZ vectors encoding D265A Fc fused with IL-2 (nucleicacid sequence: SEQ ID NO: 11; amino acid sequence: SEQ ID NO: 12),QQ6210 (nucleic acid sequence: SEQ ID NO: 13; amino acid sequence: SEQID NO: 14), E76A IL-2 (nucleic acid sequence: SEQ ID NO: 15; amino acidsequence: SEQ ID NO: 16), or E76G IL-2 (nucleic acid sequence: SEQ IDNO: 17; amino acid sequence: SEQ ID NO: 18) were co-transfected withgWIZ D265A Fc FLAG, encoding D265AFc/flag (nucleic acid sequence: SEQ IDNO: 9; amino acid sequence: SEQ ID NO: 10), into HEK293 cells using PEIin FreeStyle 293 media supplemented with OptiPro (Invitrogen). Sevendays post transfection, culture supernatants were harvested bycentrifugation (30 min at 15,000×g, 4° C.) and the supernatantsterilized by filtration through 0.22 μm filters.

Monovalent Fc/IL-2 fusions were purified by sequential TALON His-tagmetal affinity purification (Clontech) and anti-FLAG affinitychromatography (Sigma-Aldrich) following manufacturer's instructions.Elution fractions were concentrated using 15-ml 30-kDa Amicon UltraCentrifugal Devices (Millipore) and buffered exchanged into PBS. Proteinconcentration was determined by the Beer-Lambert Law:

A=εlc,

where A=absorbance at 280 nm,

-   -   β=extinction coefficient,    -   l=path length, and    -   c=concentration        Absorbance at 280 nm was measured using a NanoDrop 2000c (Thermo        Scientific). The molecular weights and extinction coefficients        of Fc/IL-2 fusion proteins were estimated from their amino acid        sequences. Fc/IL-2 fusions were secreted using HEK293 cells and        purified by sequential TALON resin and anti-FLAG affinity        chromatography.

All Fc/IL-2 fusions used in the Examples described infra all have theD265A mutation in the Fc moiety (to reduce effector function, i.e., ADCCand CDC) and are in monovalent form (to separate any effects observedfrom that caused by IL-2 bivalency) (FIG. 5). Fc/IL-2 fusions need notbe limited to the monovalent form, but can also be used in the bivalentform. The beta half-life of Fc/IL-2 is approximately 15 hours.

Example 5 Effects of Fc/IL-2 Fusions on Cell Proliferation of aCytotoxic T Cell Line

To determine the effects of CD25 binding affinity on cell proliferation,the effects of an affinity series of mouse IL-2, consisting of Fc-fusedhigh-affinity CD25-binding QQ 6.2-10 (“Fc/QQ6210”), wild-type IL-2(“Fc/IL-2”), and a non-CD25 binding IL-2 mutant named E76G (“Fc/E76G”)were tested for the ability to stimulate cell proliferation. Asdescribed supra, these three Fc/IL-2 fusions have the D265A mutation inthe Fc moiety.

Extinction coefficient, ε Molecular weight Protein (M⁻¹ cm⁻¹) (g/mol)Fc/IL-2 69870 72514.5 Fc/QQ6210 68380 72592.4 Fc/E76G 69870 72442.4

To verify that Fc/IL-2, Fc/QQ6210, Fc/E76A, and Fc/E76G were functional,they were assayed for their ability to stimulate the growth of CTLL-2cells, a murine cytotoxic T cell line. Under static conditions, allFc/IL-2 proteins support CTLL-2 growth at 100 pM, 1 nM, and 10 nM (FIG.6). The different growth kinetics resulting from stimulation withFc/E76A and Fc/E76G likely reflects the lack of CD25 binding. Forconvenience, Fc/E76G was selected for further characterization in vivo.

Example 6 Fc/IL-2 Fusions Thereof Exhibit Extended Circulation Half-LifeIn Vivo

IL-2 has a very short systemic half-life, with an initial clearancephase with an alpha half-life of 12.9 min followed by a slower phasewith a beta half-life of 85 min (Konrad et al., Cancer Res 1990;50:2009-17). Thus, one of the difficulties associated with IL-2 therapyis the maintenance of therapeutic concentrations of IL-2 (1-100 pM) fora sustained period. To this end, the in vivo circulation half-lives ofFc/IL-2, Fc/QQ6210, and Fc/E76G were determined.

Each Fc/IL-2 fusion was labeled with IRDye 800 and injectedintravenously into C57BL/6 mice as a 50 μg bolus. Blood samples werecollected over four days. Serum levels of Fc/IL-2 fusions, as determinedby the 800 nm signal within blood samples, was fitted to thebiexponential decay equation MFI(t)=Ae^(−αt)+Be^(−βt), where MFI is themean fluorescence intensity of the blood sample, t is time, and A, B, α,and β are pharmacokinetic parameters to be fitted. As shown in Table 2,all Fc/IL-2 fusions exhibit substantially prolonged in vivo persistencecompared to non-Fc fused IL-2.

TABLE 2 α β t_(1/2,α) t_(1/2,β) Protein A B (hr⁻¹) (hr⁻¹) (hr) (hr)Fc/IL-2 0.50 ± 0.15 0.70 ± 0.53 0.12 ± 0.08 0.05 ± 0.01 1.9 ± 0.9 16.4 ±3.6 Fc/QQ6210 0.44 ± 0.11 0.07 ± 0.02 0.19 ± 0.01 0.02 ± 0.00 3.6 ± 0.234.3 ± 3.2 Fc/E76G 0.71 ± 0.05 0.16 ± 0.02 0.25 ± 0.06 0.03 ± 0.00 3.0 ±0.7 25.4 ± 1.8

Example 7 Fc/IL-2 and Mutants Induce Splenomegaly and Alter T Cell andNK Cell Composition

To determine the effects of Fc/IL-2, Fc/QQ6210, and Fc/E76G on T celland NK cell composition in vivo, C57BL/6 mice were injectedintravenously once with 5 or 25 μg Fc/IL-2, Fc/QQ6210, or Fc/E76G. Fourdays later, spleens were photographed and splenocytes analyzed for T andNK cell composition by FACS.

Both doses of Fc/IL-2 fusions increased spleen size compared toPBS-treated controls (FIG. 7). With respect to CD8+ T cell and NK cellcomposition, Fc/IL-2 and Fc/QQ6210 expanded CD8+ T cell and NK cellsapproximately 2-fold, while Fc/E76G expanded these populations up to5-fold compared to PBS-treated controls (FIG. 8). The notable expansionof CD8+ T and NK cells by Fc/E76G validates the functional signaling ofthis mutant through IL-2Rβ and γ_(c).

Example 8 Toxicity of Fc/IL-2 Fusions

Total animal weight was used as a proxy for toxicity, and lung wetweight was used as an indicator for pulmonary edema and vascular leaksyndrome, which are often associated with IL-2 therapy.

As shown in FIG. 9, Fc/IL-2 and Fc/QQ6210 were well tolerated at the twodoses tested (5 μg and 25 μg), whereas Fc/E76G was highly toxic at 25μg, likely because it strongly promoted CD8+ T cell and NK cell growthas described in Example 7. Fc/E76G was well tolerated at the lower doseof 5 μg.

Fc/IL-2 fusions did not significantly affect pulmonary wet weightcompared to PBS-treated controls (FIG. 10). In contrast to a previousstudy by Krieg et al. (PNAS 2010; 107:11906-11), CD25 binding did notdrive IL-2 toxicity in the lung, as demonstrated by the similar wet lungwet weight of mice injected with all three Fc/IL-2 fusions and the PBScontrol.

Example 9 Synergistic Tumor Control by Fc/IL-2 Fusions and a TherapeuticAntibody in Melanoma

While a number of reports on combination therapy with IL-2 andtherapeutic antibodies in the treatment of cancer exist, the resultshave been largely unsuccessful, with most studies reporting no orlimited clinical benefit (Mani et al., Breast cancer research andtreatment 2009; 117; 83-9; Khan et al. Clinical Cancer Research 2006;12:7046-53). To determine whether the limited effects of previouscombination therapies could be overcome by increasing the serumhalf-life of IL-2, Fc/IL-2 fusions, in combination with a therapeuticantibody, were tested for the ability to reduce the size of establishedtumors. To this end, an art-recognized highly aggressive mouse model ofmelanoma (i.e., B16 murine melanoma) was used. This model was derivedfrom a spontaneous tumor in a C57BL/6 mouse. B16 melanoma is poorlyimmunogenic, with no MHC class II expression and very low expressionlevels of MHC class I. Moreover, the sub-line B16-F10 has beenspecifically selected for high metastatic potential.

The mouse melanoma cell line B16-F10 was a gift of Darrell J. Irvine(MIT). Cells were cultured in DMEM supplemented with FBS, L-glutamine,and pen-strep at 37° C. with 5% CO₂. At approximately 70% confluency,cells were lifted off of plates using 0.035% trypsin and EDTA. Digestionwas quenched with media and the cells pelleted by centrifugation at 1000rpm. For passaging, cells were resuspended in media to 10⁶ cells per 10ml. For tumor inoculation, cells were resuspended in PBS to 10⁶ cellsper 50 μl. Cells were verified to be mycoplasma-free by PCRamplification and staining with antibody TA99. C57BL/6 mice (The JacksonLaboratory) were used at 8 to 10 weeks of age.

On the day of tumor inoculation, mice were anesthetized with isofluraneand patches of fur were shaved from the left or right flanks to exposeskin. 1×10⁶ cells in 50 μl PBS were injected subcutaneously using a27G1/2 syringe and needle. Every other day following tumor inoculation,mice were weighed and their tumors measured using digital calipers.Tumor volumes were calculated using the modified ellipsoid volumeformula:

tumor volume=½l w ²,

wherein l=longest dimension of tumor, and w=longest dimension of tumorperpendicular to l.

Given the documented toxicity of IL-2 therapy, dosing regimens wereoptimized for the Fc/IL-2 fusions. Therapy was initiated at the time oftumor inoculation (“early treatment”) or six days after tumorinoculation, when tumor nodules were visible and palpable (“latetreatment”). 50 μg of Fc/IL-2 fusions per dose, which had previouslybeen found to be tolerable in single-dose pharmacokinetics studies, wereadministered, with subsequent doses administered every 3 days. Underthis treatment regimen, Fc/IL-2 was lethal upon the fourth or thirddose, for early and late treatment, respectively; Fc/QQ6210 waswell-tolerated; while Fc/E76G was extremely toxic, being lethal upon thesecond dose for both therapy initiation times. Toxicity was reduced whenFc/IL-2 fusions were administered at 25 μg once a week. At this dose,Fc/IL-2 fusions were well tolerated. The results of these toxicitystudies, and the resulting dosing regimen, is analogous to similarexperiments reported previously with PEG-IL-2 (Zimmerman et al., CancerResearch 1989; 49:6521-8).

Antibody TA99 was used as the therapeutic antibody for the combinationtherapy. TA99 is a murine IgG2a antibody that recognizes the melanosomeantigen tyrosinase-related protein-1 (Tyrp-1) and is of identicalspecificity as an antibody originally isolated from the serum of amelanoma patient (Houghton et al., Prog Clin Biol Res 1983;119:199-205). Tolerable and effective dosing regiments were alsodetermined for Fc/IL-2 fusions in combination with 100 μg antibody TA99.As described above, 25 μg Fc/E76G with TA99 once every three days waslethal upon the second dose, while reducing the frequency to once weeklywas well-tolerated.

Having established a tolerable dosing regimen for Fc/IL-2 fusions withTA99, the efficacy of Fc/IL-2 fusions alone or in combination with TA99were tested for controlling subcutaneous B16-F10 tumors. C57BL/6 mice(n=5/group) were injected subcutaneously with B16-F10 melanoma cells andtumors were allowed to establish for six days. With tumor nodulesvisible and palpable, mice were treated with 25 μg Fc/IL-2 or 25 μgFc/IL-2 in combination with 100 μg TA99, with subsequent dosesadministered every six days. As controls, B16-F10 tumor-bearing micewere treated with PBS, 6 μg free IL-2 (this corresponds to astoichiometrically equivalent amount to each of the Fc/IL-2 fusionsused), 100 μg TA99, or 6 μg free IL-2 with 100 μg TA99.

As expected for B16-F10 tumors, all PBS-treated mice required euthanasiadue to tumor size within two weeks of tumor inoculation. Consistent withprevious reports, IL-2 or TA99 alone was insufficient to delay B16-F10growth (FIG. 11). IL-2 in combination with TA99 delayed tumorprogression to a limited degree in some mice (FIG. 11). However, alltumors in mice treated with IL-2 and TA99 reached euthanasia criteriawithin the course of therapy. Fc/IL-2 alone, by contrast, significantlyreduced tumor burden, and Fc/IL-2 in combination with TA99 exertedsynergistic tumor control, as evidenced by the complete lack of tumorgrowth in 4 of 5 mice over the course of 30 days (FIG. 11). Relative toPBS-treated controls, a single dose of Fc/IL-2 reduced average tumorvolume on day 12 by 50%; a single treatment with Fc/IL-2 in combinationwith TA99 reduced average tumor volume on day 12 by 83%. Continuingtherapy with Fc/IL-2 delayed tumors from reaching a volume of 500 mm³ by7 days. For Fc/IL-2 and TA99 combination therapy, four of five tumorsremained below 85 mm³ during treatment. Only one of five tumorsprogressed to reach 500 mm³, although growth was significantly delayedby 16.7 days relative to PBS-treated controls. This series of treatmentswas also plotted as average tumor volumes for each group with standarddeviations (FIG. 12). These data confirm the limited efficacy of IL-2and TA99 alone compared to Fc/IL2 alone, and confirm the synergisticeffect conferred by the Fc/IL-2+TA99 group compared to Fc/IL or TA99alone, and the marked survival benefit conferred by the Fc/IL-2 and TA99combination therapy over IL-2 and TA99 combination therapy. Statisticsare shown below.

TABLE 3 P values for Fc/IL-2 + TA99 treatment vs. indicated groups atdifferent days after tumor inoculation (unpaired t-test using data fromFIG. 12) Days after turmor inoculation PBS IL-2 Fc/IL-2 TA99 IL-2 + TA9910 0.0038 0.0243 0.1190 0.0204 0.1588 12 0.0047 0.0174 0.1387 0.00230.1879 14 0.1414 0.0702 17 0.1678 0.0407 18 0.0385

The synergistic control of Fc/IL-2 in combination with TA99 was furtherdemonstrated when the data were plotted as the number of days it tookfor tumor area to become >100 mm². Euthanasia criteria was tumorarea >100 mm², and tumor area was calculated as l×w, wherein l=longestdimension of the tumor and w=longest dimension perpendicular to 1. Asshown in FIG. 13, while Fc/IL-2 treated mice took somewhat longer toachieve a tumor area >100 mm² than PBS-treated controls, IL-2, TA99, andIL2+TA99 groups, the duration required for tumors in the Fc/IL-2+TA99group to achieve a tumor area >100 mm² was more than double that of anyof the other groups (n=5 mice/group) (Table 4).

TABLE 4 Average number of days (±standard deviation) to tumor area >100mm² PBS Fc/IL-2 TA99 FC/IL-2 + TA99 IL-2 IL-2 + TA99 13.00 ± 0.92 20.13± 6.56 16.35 ± 2.31 46.18 ± 8.61 12.94 ± 1.50 16.83 ± 5.58These data further support the synergistic tumor controlling effectexerted by Fc/IL-2 and TA99 combination therapy, as well as thesynergistic effect of the combination therapy on prolonging survival.

The weight of animals treated with all therapies tracked withPBS-treated controls, suggesting these therapies were well-tolerated bythe mice (FIG. 14). These findings collectively demonstrate that whileFc/IL-2 can delay tumor progression, the combination of Fc/IL-2 withTA99 exerted synergistic tumor control and potently suppressed tumorgrowth. Moreover, the results also indicate that Fc/IL-2 and TA99combination therapy increased lifespan.

Example 10 Separate Administration of Fc/IL-2 and TA99 RetainsTherapeutic Efficacy

To determine whether simultaneous administration of Fc/IL-2 and TA99 isrequired or optimal for therapeutic efficacy, the administration of thetwo agents were separated by intervals ranging from 0 to 3 days. C57BL/6mice were injected subcutaneously with 10⁶ B16-F10 melanoma cells, andtumors were allowed to establish for 6 days. With tumor nodules visibleand palpable, mice were treated with a single dose each of 25 μg Fc/IL-2and 100 μg TA99, separated by approximately 0, 6, 12, 18, 24, 48, or 72hours.

While none of these single-dose treatments cured the mice, all delayedexponential tumor growth. To compare their efficacy quantitatively, wedevised the metric 4V₀, representing the time for tumor volume to doubletwice (FIG. 15). This quantity captures the delayed growth due totreatment as well as the variability in initial tumor volume. Separatingadministration of the two agents by up to two days did not significantlyaffect efficacy, while separating the two agents by three days reducedtumor control effects. These data suggest that Fc/IL-2 and TA99 do notneed to be administered simultaneously, but can be separated by up to 3days and retain tumor controlling effects.

Example 11 Fc/IL-2 in Combination with TA99 Promotes LymphocyteRecruitment to the Periphery of Tumors

The effects of combination therapy with Fc/IL-2 and the TA99 antibody onlymphocyte recruitment to the periphery of tumors were assessed. C57BL/6mice were injected subcutaneously with B16-F10 melanoma cells (1×10⁶cells) and tumors were allowed to establish for 8 days. Mice were thenadministered either PBS or 25 μg of Fc/IL-2 and 100 μg of TA99 antibody.Four days after single-dose therapy, tumor tissue was harvested, fixedin formalin, and stained with hematoxylin and eosin. As shown in FIG.16, Fc/IL-2+TA99 showed significant recruitment of lymphocytes to theperiphery of tumors, whereas only few lymphocytes were observed in theperiphery of tumors in PBS treated mice.

Example 12 Combination Therapy of Fc/IL-2 and TA99 Suppresses SecondaryTumor Formation

While combination therapy with Fc/IL-2 and TA99 effectively controlstumor growth as discussed supra, it is unclear whether the combinationtherapy promotes an anti-tumor memory response. Protection against asecondary tumor challenge reflects the development of a systemicanti-tumor immune response, which would also indicate a potentialprotective effect against tumor metastasis. To address this possibility,C57BL/6 mice were injected subcutaneously with B16-F10 melanoma cells(1×10⁵) and tumors were allowed to establish for 6 days. After fivedoses of Fc/IL-2 (25 μg) and TA99 (100 μg), treatment was stopped toassess the duration of the therapeutic effect and the development of anyanti-tumor memory responses.

Twelve days after the last treatment, corresponding to 42 days aftertumor inoculation, the three remaining mice of this treatment group werere-challenged with 10⁵ B16-F10 cells subcutaneously in the oppositeflank. Two naive C57BL/6 mice were similarly inoculated with 10⁵ B16-F10cells as controls. All three primary tumors eventually enteredexponential growth and required euthanasia by day 26 after the lasttreatment. However, at the time of euthanasia, 14 days afterre-challenge, two of three Fc/IL-2 and TA99 treated mice remainedtumor-free at the secondary challenge site (FIG. 17). These data suggestthat Fc/IL-2 and TA99 combination therapy induced an anti-tumor memoryresponse and prevented the establishment of new tumors upon re-challengewith cancer cells.

Example 13 CD25 Binding Affinity Required for Maximal Therapeutic Effectof Combination Therapy with Fc/IL-2 and TA99

To determine whether CD25 binding affinity is required for the efficacyof combination therapy with Fc/IL-22 and TA99, Fc/IL-2 fusions weretested for their tumor controlling effects. C57BL/6 mice were injectedsubcutaneously with B16-F10 melanoma cells (1×10⁶) and tumors wereallowed to establish for six days. With tumor nodules visible andpalpable, mice were treated with 25 μg Fc/QQ610 (high affinityCD25-binding IL-2) or 25 μg Fc/E76G (non-CD25 binding IL-2), alone or incombination with 100 μg TA99, with subsequent doses administered everysix days.

Similar to Fc/IL-2, both Fc/QQ6210 and Fc/E76G alone delayed tumorprogression compared to PBS-treated controls and IL-2 (FIGS. 11 and 18),although mice treated with Fc/E76G alone reached euthanasia criteriawithin the course of therapy. When combined with TA99, Fc/QQ6210 exertedsimilar tumor growth suppressing effects than Fc/IL-2, whereas tumorsresumed exponential growth with Fc/E76G+TA99 (FIG. 18). These datasuggest that CD25 binding is required for the full therapeutic efficacyof the Fc/IL-2 and TA99 combination therapy, and that CD25+ cells arekey effectors of the Fc/IL-2 effect.

Example 14 CD8+ T Cells and NK Cells Contribute to Therapeutic Effect

Selective cell depletion was performed to determine which cell typeswere responsible for the therapeutic efficacy of the Fc/IL-2 and TA99combination therapy. NK and CD8+ T cells were likely candidates, giventheir ability to mediate ADCC, cytotoxic abilities, and receivestimulation by IL-2. Anti-NK1.1 antibody (Bio X Cell, West Lebanon,N.H.; clone PK136) was used to deplete NK and NKT cells, and anti-CD8aantibody (Bio X Cell, West Lebanon, N.H.; clone 2.43) was used todeplete CD8+ cells. C57 BL/6 mice (n=5/group) were injectedsubcutaneously with 10⁶ B16F10 melanoma cells. Four days post tumorinoculation, and every 4 days afterward, 400 μg of antibody in 100 μlPBS were injected intra-peritoneally. Cell depletion was confirmed byflow cytometry of splenocytes (FIG. 19). Six days after tumorinoculation, mice were injected intravenously with PBS or 25 μg Fc/IL-2and 100 μg TA99. Subsequent doses were administered every 6 days.

As shown in FIG. 20, the suppressive effects of Fc/IL-2 and TA99combination therapy were lost in the absence of CD8+ T cells, suggestingthat CD8+ T cells are required and are a major component of the efficacyof this combination therapy. Moreover, depletion of NK cells partiallydecreased tumor progression, suggesting that NK cells also play a rolein the efficacy of this combination therapy. These findings are incontrast to notion that therapeutic antibodies exert their effectsmainly via ADCC by NK cells.

Example 15 Tumor Control by Fc/IL-2 Fusions and a Therapeutic Antibodyin Colon Cancer

Given the efficacy of the Fc/IL-2 and TA99 combination therapy, it islikely that Fc/IL-2 would synergize with other anti-tumor antibodies aswell. Thus, the generalizability of the pronounced effects ofextended-PK IL-2 and therapeutic antibody combination therapy were alsotested in a mouse model of colon cancer.

MC38-CEA cells (1×10⁶), a colon cancer cell line, were injected into theflank of four C57 BL/6 J mice to induce tumor establishment. Fc-IL-2 wasinjected at 25 μg/mouse retroorbitally. Sm3E anti-CEA antibody, ahumanized IgG1 antibody with extremely high affinity for soluble andmembrane-bound CEA (K_(D)=20 pM) (Graff et al., Protein Eng Des Sel2004; 17:293-304), was injected at a dosage of 200 μg/mouseretroorbitally as shown in FIG. 21. Tumor volume was assessed asdescribed in Example 7.

Pronounced decreases in tumor volume were observed in mice immediatelyafter injection of Fc/IL-2 and the sm3e anti-CEA antibody (FIG. 21).Notably, tumors were eradicated in mouse 2 and 3 upon repeated or singlecombination therapy, respectively. Mouse 1 was euthanized at day 26 dueto an ulcerated tumor. Mouse 2 was injected again with MC38-CEA cells inthe left flank on day 24 after primary tumor implantation, but nosubsequent tumors were formed. These findings demonstrate thegeneralizability of the pronounced therapeutic effects exerted by thecombination of an extended-PK IL-2 and therapeutic antibody.

Example 16 Tumor Control by Other Types of Extended-PK IL-2 andTherapeutic Antibodies

Bivalent Fc/IL-2, PEG-IL-2, HSA-IL-2, Fn3(HSA)-IL-2 in combination withTA99 antibody or sm3e anti-CEA antibody are tested for tumor controllingeffects as described in Examples 9 and 15, respectively.

Example 17 Control of Other Types of Cancer with Extended-PK IL-2 andTherapeutic Agents

Fc-IL-2, PEG-IL-2, HSA-IL-2, and Fn3(HSA)-IL-2 are tested in other mousemodels of cancer in combination with therapeutic antibodies that targetthe particular cancer being tested. Various mouse models of cancer areknown in the art. For example, Kras^(LSL-G12D/+); p53^(Flox/Flox) (KP)mice (“KP mice”) (Xue et al., Cancer Discovery 2011; 1:236-47; Winslowet al., Nature 2011; 473; 101-4) can be made to express human CEA as asurface antigen using Cre recombinase vector, allowing for testing theefficacy of a combination therapy comprising extended-PK IL-2 and ananti-CEA antibody. The KP mice can also be used to test the efficacy ofa combination therapy comprising extended-PK IL-2 and a therapeuticagent, such as the small molecule inhibitors gefitinib and erlotinib.

TABLE 5 SEQUENCES SEQ ID NO DESCRIPTION SEQUENCE  1 Mouse IL-2GCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAG (nucleic acidCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAG sequence)GAGCTCCTGAGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGAACTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATC TCAACAAGCCCTCAA 2 Mouse IL-2 APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRML(amino acid TFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVsequence) VKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ  3 QQ6210GCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAACAGCAGCAG (nucleic acidCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAG sequence)GAACTCCTGAGTAGGATGGAGGATCACAGGAACCTGAGACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCGAGCAGGCCACAGAATTGGAAGATCTTCAGTGCCTAGAAGATGAACTTGAACCACTGCGGCAAGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGACGATGAGCCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATC TCAACAAGCCCTCAA 4 QQ6210 APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEDHRNLRLPRML(amino acid TFKFYLPEQATELEDLQCLEDELEPLRQVLDLTQSKSFQLEDAENFISNIRVTVsequence) VKLKGSDNTFECQFDDEPATVVDFLRRWIAFCQSIISTSPQ  5 E76AGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAG (nucleic acidCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAG sequence)GAGCTCCTGAGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGCTCTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATC TCAACAAGCCCTCAA 6 E76A APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRML(amino acid TFKFYLPKQATELKDLQCLEDALGPLRHVLDLTQSKSFQLEDAENFISNIRVTVsequence) VKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ  7 E76GGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAG (nucleic acidCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAG sequence)GAGCTCCTGAGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGGTCTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATC TCAACAAGCCCTCAA 8 E76G APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRML(amino acid TFKFYLPKQATELKDLQCLEDGLGPLRHVLDLTQSKSFQLEDAENFISNIRVTVsequence) VKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ  9 D265A Fc/FlagATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCA (nucleicCGATGTGAGCCCAGAGTGCCCATAACACAGAACCCCTGTCCTCCACTCAAAGAG acid sequence)TGTCCCCCATGCGCAGCTCCAGACCTCTTGGGTGGACCATCCGTCTTCATCTTC (C-terminal flagCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTtag is underlined)GTGGTGGTGGCCGTGAGCGAGGATGACCCAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGCCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAAGGTGGCGGATCTGACTACAAGGACGACGATGACAAG TGATAA 10D265A Fc/Flag MRVPAQLLGLLLLWLPGARCEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIF(amino acid PPKIKDVLMISLSPMVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNsequence) STLRVVSALPIQHQDWMSGKEEKCKVNNRALPSPIEKTISKPRGPVRAPQVYVL(C-terminal flag tagPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYF is underlined)MYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGKGGGSDYKDDDDK 11D265A Fc/wt mIL-2 ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCA(nucleic acid CGATGTGAGCCCAGAGTGCCCATAACACAGAACCCCTGTCCTCCACTCAAAGAGsequence) TGTCCCCCATGCGCAGCTCCAGACCTCTTGGGTGGACCATCCGTCTTCATCTTC(C-terminal 6x hisCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTtag is underlined)GTGGTGGTGGCCGTGAGCGAGGATGACCCAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGCCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAAGGAGGGGGCTCCGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGAACTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAACACCATCAC CACCATCACTGATAA12 D265A Fc/wt mIL-2MRVPAQLLGLLLLWLPGARCEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIF (amino acidPPKIKDVLMISLSPMVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYN sequence)STLRVVSALPIQHQDWMSGKEEKCKVNNRALPSPIEKTISKPRGPVRAPQVYVL(C-terminal 6x hisPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFtag is underlined)MYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGKGGGSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQHHHHHH** 13 D265A Fc/QQ6210ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCA (nucleic acidCGATGTGAGCCCAGAGTGCCCATAACACAGAACCCCTGTCCTCCACTCAAAGAG sequence)TGTCCCCCATGCGCAGCTCCAGACCTCTTGGGTGGACCATCCGTCTTCATCTTC(C-terminal 6x hisCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTtag is underlined)GTGGTGGTGGCCGTGAGCGAGGATGACCCAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGCCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAAGGAGGGGGCTCCGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAACTCCTGAGTAGGATGGAGGATCACAGGAACCTGAGACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCGAGCAGGCCACAGAATTGGAAGATCTTCAGTGCCTAGAAGATGAACTTGAACCACTGCGGCAAGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGACGATGAGCCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAACACCATCAC CACCATCACTGATAA14 D265A Fc/QQ6210MRVPAQLLGLLLLWLPGARCEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIF (amino acidPPKIKDVLMISLSPMVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYN sequence)STLRVVSALPIQHQDWMSGKEEKCKVNNRALPSPIEKTISKPRGPVRAPQVYVL(C-terminal 6x hisPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFtag is underlined)MYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGKGGGSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEDHRNLRLPRMLTFKFYLPEQATELEDLQCLEDELEPLRQVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDEPATVVDFLRRWIAFCQSIIHSTSPQHHHHHH 15 D265A Fc/E76AATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCA (nucleic acidCGATGTGAGCCCAGAGTGCCCATAACACAGAACCCCTGTCCTCCACTCAAAGAG sequence)TGTCCCCCATGCGCAGCTCCAGACCTCTTGGGTGGACCATCCGTCTTCATCTTC(C-terminal 6x hisCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTtag is underlined)GTGGTGGTGGCCGTGAGCGAGGATGACCCAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGCCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAAGGAGGGGGCTCCGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGCTCTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAACACCATCAC CACCATCACTGATAA16 D265A Fc/E76A MRVPAQLLGLLLLWLPGARCEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIF(amino acid PPKIKDVLMISLSPMVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNsequence) STLRVVSALPIQHQDWMSGKEEKCKVNNRALPSPIEKTISKPRGPVRAPQVYVL(C-terminal 6x hisPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFtag is underlined)MYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGKGGGSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDALGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIIHSTSPQHHHHHH 17 D265A Fc/E76GATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCA (nucleic acidCGATGTGAGCCCAGAGTGCCCATAACACAGAACCCCTGTCCTCCACTCAAAGAG sequence)TGTCCCCCATGCGCAGCTCCAGACCTCTTGGGTGGACCATCCGTCTTCATCTTC(C-terminal 6x hisCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTtag is underlined)GTGGTGGTGGCCGTGAGCGAGGATGACCCAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGCCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAAGGAGGGGGCTCCGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGGTCTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAACACCATCAC CACCATCACTGATAA18 D265A Fc/E76G MRVPAQLLGLLLLWLPGARCEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIF(amino acid PPKIKDVLMISLSPMVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNsequence) STLRVVSALPIQHQDWMSGKEEKCKVNNRALPSPIEKTISKPRGPVRAPQVYVL(C-terminal 6x hisPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFtag is underlined)MYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGKGGGSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDGLGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQHHHHHH 19 mIL-2 QQ 6.2-4GCACCCACCTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAACAGCAGCAG (nucleic acidCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAG sequence)GAGCTCCTGAGCAGGATGGAGGATTCCAGGAACCTGAGACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGGAAGATCTTCAGTGCCTAGAAGATGAACTTGAACCTCTGCGGCAAGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGCCAGCAACTGTGGTGGGCTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATC TCAACGAGCCCTCAA20 mIL-2 QQ 6.2-4 APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEDSRNLRLPRML(amino acid TFKFYLPKQATELEDLQCLEDELEPLRQVLDLTQSKSFQLEDAENFISNIRVTVsequence) VKLKGSDNTFECQFDDEPATVVGFLRRWIAFCQSIISTSPQ 21 mIL-2 QQ 6.2-8GCACCCACCTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAACAGCAGCAG (nucleic acidCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGT sequence)AGGATGGAGGATCACAGGAACCTGAGACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGGAAGATCTTCAGTGCCTAGAAGATGAACTTGAACCTCTGCGGCAAGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGCCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCT CGA 22mIL-2 QQ 6.2-8 APTSSSTSSSTAEAQQQQQQQQHLEQLLMDLQELLSRMEDHRNLRLPRMLTFKF(amino acid YLPKQATELEDLQCLEDELEPLRQVLDLTQSKSFQLEDAENFISNIRVTVVKLKsequence) GSDNTFECQFDDEPATVVDFLRRWIAFCQSIISTSPR 23 mIL-2 QQ 6.2-10GCACCCACCTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAACAGCAGCAG (nucleic acidCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAG sequence)GAACTCCTGAGTAGGATGGAGGATCACAGGAACCTGAGACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCGAGCAGGCCACAGAATTGGAAGATCTTCAGTGCCTAGAAGATGAACTTGAACCACTGCGGCAAGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGACGATGAGCCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATC TCAACAAGCCCTCAG24 mIL-2 QQ 6.2-10APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEDHRNLRLPRML (amino acidTFKFYLPEQATELEDLQCLEDELEPLRQVLDLTQSKSFQLEDAENFISNIRVTV sequence)VKLKGSDNTFECQFDDEPATVVDFLRRWIAFCQSIISTSPQ 25 mIL-2 QQ 6.2-11GCACCCACCTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAACAGCAGCAG (nucleic acidCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTG sequence)AGCAGGATGGAGGATTCCAGGAACCTGAGACTCCCCAGAATGCTCACCTTCAAATTTTACTTGCCCGAGCAGGCCACAGAATTGAAAGATCTCCAGTGCCTAGAAGATGAACTTGAACCTCTGCGGCAAGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGACGATGAGCCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGC CCTCAG 26mIL-2 QQ 6.2-11 APTSSSTSSSTAEAQQQQQQQQQHLEQLLMDLQELLSRMEDSRNLRLPRMLTFK(amino acid FYLPEQATELKDLQCLEDELEPLRQVLDLTQSKSFQLEDAENFISNIRVTVVKLsequence) KGSDNTFECQFDDEPATVVDFLRRWIAFCQSIISTSPQ 27 mIL-2 QQ 6.2-13GCACCCACCTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAACAGCAGCAG (nucleic acidCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAG sequence)GAGCTCCTGAGTAGGATGGAGGATCACAGGAACCTGAGACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCGAGCAGGCCACAGAATTGAAAGATCTCCAGTGCCTAGAAGATGAACTTGAACCTCTGCGGCAGGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGCCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATC TCAACAAGCCCTCAG28 mIL-2 QQ 6.2-13APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEDHRNLRLPRML (amino acidTFKFYLPEQATELKDLQCLEDELEPLRQVLDLTQSKSFQLEDAENFISNIRVTV sequence)VKLKGSDNTFECQFDDEPATVVDFLRRWIAFCQSIISTSPQ 29 Full length humanATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACA IL-2 (nucleicAACAGTGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCAT acid sequence)TTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTGACTTGA 30 Full length humanMYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP IL-2 (aminoKLTRMLTEKEYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNEHLRPRDLISN acid sequence)INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 31 Human IL-2 withoutGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTG signal peptideCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTC (nucleic acidACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAA sequence)CATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTGACTTGA 32 Human IL-2 withoutAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELK signal peptideHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYA (amino acidDETATIVEFLNRWITFCQSIISTLT sequence) 33 Human IgG1 constantASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPregion (amino acidAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT sequence)CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK 34Human IgG1 Fc EPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEdomain (amino DPEVKLNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVacid sequence) SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK

1. A method for treating cancer in a subject comprising administering anextended-pharmacokinetic (PK) interleukin (IL)-2, and a therapeuticagent in an amount effective to treat cancer.
 2. The method of claim 1,wherein the extended-PK IL-2 comprises a fusion protein.
 3. The methodof claim 2, wherein the fusion protein comprises an IL-2 moiety and amoiety selected from the group consisting of an immunoglobulin fragment,human serum albumin, and Fn3.
 4. The method of claim 1, wherein theextended-PK IL-2 comprises an IL-2 moiety conjugated to a non-proteinpolymer.
 5. The method of claim 4, wherein the non-protein polymer ispolyethylene glycol.
 6. The method of claim 3, wherein the fusionprotein comprises an IL-2 moiety and an Fc domain.
 7. The method ofclaim 6, wherein the Fc domain is mutated to reduce binding to Fcγreceptors, complement proteins, or both.
 8. The method of claim 7,wherein the fusion protein comprises a monomer of one IL-2 moiety linkedto an Fc domain as a heterodimer.
 9. The method of claim 7, wherein thefusion protein comprises a dimer of two IL-2 moieties linked to an Fcdomain as a heterodimer.
 10. The method of any one of claims 1-9,wherein the IL-2 is mutated such that it has higher affinity for theIL-2R alpha receptor compared to unmodified IL-2.
 11. The method of anyone of claims 1-10, wherein the therapeutic agent is selected from thegroup consisting of a therapeutic antibody, a therapeutic protein, asmall molecule, an antigen, and a population of cells.
 12. The method ofclaim 11, wherein the therapeutic agent is a therapeutic antibody. 13.The method of any one of claims 1-12, wherein the extended-PK IL-2 andthe therapeutic agent are administered simultaneously or sequentially.14. The method of any one of claims 1-12, wherein the extended-PK IL-2and the therapeutic agent are administered within three days of eachother.
 15. The method of any one of claims 1-14, wherein the cancer isselected from the group consisting of melanoma, colon cancer, breastcancer, renal cancer, testicular cancer, ovarian cancer, prostatecancer, cancer of the small intestine, cancer of the esophagus, cervicalcancer, lung cancer, lymphoma, and leukemia.
 16. The method of any oneof claims 1-15, further comprising administering an additionaltherapeutic agent selected from the group consisting of a cytokine, achemotherapeutic agent and a population of cytotoxic T cells.
 17. Amethod of inhibiting growth and/or proliferation of tumor cells in asubject comprising administering an extended-PK interleukin (IL)-2 and atherapeutic antibody in an amount effective to inhibit growth and/orproliferation of tumor cells in the subject.
 18. A method of reducingtumor size in a subject comprising administering an extended-PK IL-2 anda therapeutic antibody in an amount effective to reduce tumor size inthe subject.
 19. The method of claim 18, wherein the tumor volume isreduced by at least 30%.
 20. The method of claim 19, wherein the tumorvolume is reduced at least 50%.
 21. The method of claim 20, wherein thetumor volume is reduced at least 80%.
 22. The method of claim 21,wherein the tumor volume is reduced at least 90%.
 23. The method ofclaim 18, wherein the extended-PK IL-2 and therapeutic antibody reducestumor size to a greater extent than achieved by a combination of IL-2and a therapeutic antibody.
 24. A method of increasing recruitment oflymphocytes to the periphery of a tumor in a subject comprisingadministering an extended-PK IL-2 and a therapeutic antibody in anamount effect to increase recruitment of lymphocytes to the periphery ofthe tumor.
 25. A method of inhibiting metastases of a primary tumor in asubject with an established primary tumor comprising administering anextended-PK IL-2 and a therapeutic antibody in an amount effective toinhibit metastases in the subject.
 26. A method of increasing IL-2R betaand IL-2R gamma signaling in a lymphocyte in vivo comprisingadministering an extended-PK IL-2 and a therapeutic antibody to the cellin an amount effective to increase IL-2R beta and IL-2R gamma signaling.27. A method of prolonging survival of a subject with a tumor comprisingadministering an extended-PK IL-2, and a therapeutic antibody in anamount effective to prolong survival in the subject.
 28. The method ofclaim 25, wherein the subject is a mouse model of cancer and survival isprolonged by 25 days or more.
 29. A method of stimulating T cells and/orNK cells in a subject comprising administering an extended-PK IL-2, anda therapeutic antibody in an amount effective to stimulate T cellsand/or NK cells in a subject.
 30. A method of enhancingantibody-dependent cell-mediated cytotoxicity (ADCC) and/or cytotoxic Tlymphocyte (CTL) responses in a subject comprising administering anextended-PK IL-2, and a therapeutic antibody in an amount effective toenhance ADCC and/or CTL in the subject.
 31. A method of increasing thenumber of CD8+ T cells in a subject comprising administering anextended-PK IL-2, and a therapeutic antibody in an amount effective toincrease the number of CD8+ T cells in the subject.
 32. A method oftreating cancer in a subject comprising administering an extended-PKIL-2, and a therapeutic antibody in an amount effective to treat cancer.33. The method of any one of claims 17-32, wherein the extended-PK IL-2comprises a fusion protein.
 34. The method of claim 33, wherein thefusion protein comprises an IL-2 moiety and a moiety selected from thegroup consisting of an immunoglobulin fragment, human serum albumin, andFn3.
 35. The method of any one of claims 17-32, wherein the extended-PKIL-2 comprises an IL-2 moiety conjugated to a non-protein polymer. 36.The method of claim 35, wherein the non-protein polymer is polyethyleneglycol.
 37. The method of claim 33, wherein the fusion protein comprisesan IL-2 moiety and an Fc domain.
 38. The method of claim 37, wherein theFc domain is modified to reduce binding to Fcγ receptors, complementproteins, or both.
 39. The method of claim 31, wherein the fusionprotein comprises a monomer of one IL-2 moiety linked to an Fc domain.40. The method of claim 31, wherein the fusion protein comprises a dimerof two IL-2 moieties linked to an Fc domain as a heterodimer.
 41. Themethod of any one of claims 17-38, wherein the IL-2 is mutated such thatit has higher affinity for the IL-2R alpha receptor compared tounmodified IL-2.